section 2.6 nonclinical summary section 2.6.6 toxicology … · 2015-05-29 · toxicology...

SECTION 2.6 NONCLINICAL SUMMARY

Section 2.6.6 Toxicology Written Summary

EMTRICITABINE/RILPIVIRINE/ TENOFOVIR DISOPROXIL FUMARATE

FIXED-DOSE COMBINATION

Gilead Sciences International Limited

17AUG2010

CONFIDENTIAL AND PROPRIETARY INFORMATION

Emtricitabine/Rilpivirine/Tenofovir Disoproxil Fumarate Section 2.6.6 Toxicology Written Summary Final

CONFIDENTIAL 17AUG2010

TABLE OF CONTENTS

SECTION 2.6 NONCLINICAL SUMMARY.......................................................................................................1 TABLE OF CONTENTS .......................................................................................................................................2 GLOSSARY OF ABBREVIATIONS AND DEFINITION OF TERMS...............................................................5 PHARMACOKINETIC ABBREVIATIONS.........................................................................................................8 2.6. NONCLINICAL SUMMARY.......................................................................................................................9

2.6.6. TOXICOLOGY WRITTEN SUMMARY ........................................................................................9 2.6.6.1. Brief Summary ................................................................................................................9 2.6.6.2. Single-Dose Toxicity.....................................................................................................19

2.6.6.2.1. Emtricitabine: Mouse...............................................................................19 2.6.6.2.2. Rilpivirine: Mouse ...................................................................................19 2.6.6.2.3. Emtricitabine: Rat ....................................................................................20 2.6.6.2.4. Rilpivirine: Rat.........................................................................................20 2.6.6.2.5. Tenofovir DF: Rat....................................................................................21 2.6.6.2.6. Rilpivirine: Dog .......................................................................................21 2.6.6.2.7. Tenofovir DF: Dog ..................................................................................22 2.6.6.2.8. Emtricitabine/Rilpivirine/Tenofovir DF...................................................22

2.6.6.3. Repeat-Dose Toxicity....................................................................................................23 2.6.6.3.1. Mouse ......................................................................................................23 2.6.6.3.2. Rat............................................................................................................32 2.6.6.3.3. Rabbit.......................................................................................................49 2.6.6.3.4. Dog ..........................................................................................................49 2.6.6.3.5. Monkey ....................................................................................................68 2.6.6.3.6. Emtricitabine/Rilpivirine/Tenofovir DF...................................................72

2.6.6.4. Genotoxicity..................................................................................................................74 2.6.6.4.1. In Vitro Studies ........................................................................................74 2.6.6.4.2. In Vivo Studies ........................................................................................79 2.6.6.4.3. Emtricitabine/Tenofovir DF.....................................................................83 2.6.6.4.4. Emtricitabine/Rilpivirine/Tenofovir DF...................................................84

2.6.6.5. Carcinogenicity .............................................................................................................84 2.6.6.5.1. Emtricitabine: TP-0006: 2-Year Oral Oncogenicity Study in CD-1

Mice .........................................................................................................84 2.6.6.5.2. Emtricitabine: TP-0006: 2-Year Oral Oncogenicity Study in CD Rats....85 2.6.6.5.3. Rilpivirine: 2-Year Carcinogenicity Study in Mice..................................86 2.6.6.5.4. Rilpivirine: 2-Year Carcinogenicity Study in Rats...................................89 2.6.6.5.5. Tenofovir DF: An Oral Carcinogenicity Study of Tenofovir DF in the

Albino Mouse...........................................................................................93 2.6.6.5.6. Tenofovir DF: An Oral Carcinogenicity Study of Tenofovir DF in the

Albino Rat................................................................................................95 2.6.6.5.7. Emtricitabine/Rilpivirine/Tenofovir DF...................................................96

2.6.6.6. Reproductive and Developmental Toxicity...................................................................97 2.6.6.6.1. Fertility and Early Embryonic Development............................................97 2.6.6.6.2. Embryo-Fetal Development ...................................................................101 2.6.6.6.3. Prenatal and Postnatal Development Including Maternal Function .......111 2.6.6.6.4. Emtricitabine/Rilpivirine/Tenofovir DF.................................................119

2.6.6.7. Local Tolerance...........................................................................................................119 2.6.6.7.1. Rilpivirine: Eye Irritation – Bovine Corneal Opacity-Permeability

Assay......................................................................................................119 2.6.6.7.2. Rilpivirine: Phototoxicity – Balb/c Mouse 3T3 Fibroblast Assay..........120 2.6.6.7.3. Rilpivirine: Skin Irritation – In Vivo......................................................120 2.6.6.7.4. Tenofovir DF: A Primary Eye Irritation Study in Rabbits with

Tenofovir DF .........................................................................................120



2.6.6.7.5. Tenofovir DF: A Primary Skin Irritation Study in Rabbits with Tenofovir DF .........................................................................................121

2.6.6.7.6. Emtricitabine/Rilpivirine/Tenofovir DF.................................................121 2.6.6.8. Other Toxicity Studies.................................................................................................122

2.6.6.8.1. Antigenicity............................................................................................122 2.6.6.8.2. Immunotoxicity ......................................................................................123 2.6.6.8.3. Mechanistic Studies ...............................................................................124 2.6.6.8.4. Dependence............................................................................................133 2.6.6.8.5. Studies on Metabolites ...........................................................................133 2.6.6.8.6. Studies on Impurities .............................................................................133 2.6.6.8.7. Qualification of Impurities .....................................................................141 2.6.6.8.8. Other Studies..........................................................................................150

2.6.6.9. Discussion and Conclusions ........................................................................................152 2.6.6.9.1. Target Organ Toxicity: Emtricitabine and Tenofovir DF ......................152 2.6.6.9.2. Target Organ Toxicity: Rilpivirine ........................................................153 2.6.6.9.3. Target Organ Toxicity: Emtricitabine/Rilpivirine/Tenofovir DF...........159 2.6.6.9.4. Genetic Toxicology: Emtricitabine and Tenofovir DF...........................159 2.6.6.9.5. Genetic Toxicology: Rilpivirine.............................................................160 2.6.6.9.6. Genetic Toxicology: Emtricitabine/Rilpivirine/Tenofovir DF...............161 2.6.6.9.7. Carcinogenicity: Emtricitabine and Tenofovir DF.................................161 2.6.6.9.8. Carcinogenicity: Rilpivirine...................................................................161 2.6.6.9.9. Carcinogenicity: Emtricitabine/Rilpivirine/Tenofovir DF .....................162 2.6.6.9.10. Reproductive Toxicity: Emtricitabine and Tenofovir DF ......................163 2.6.6.9.11. Reproductive Toxicity: Rilpivirine ........................................................163 2.6.6.9.12. Reproductive Toxicity: Emtricitabine/Rilpivirine/Tenofovir DF...........163 2.6.6.9.13. Metabolites/Impurities: Emtricitabine/Tenofovir DF.............................163 2.6.6.9.14. Metabolites/Impurities: Rilpivirine ........................................................164 2.6.6.9.15. Metabolites/Impurities: Emtricitabine/Rilpivirine/Tenofovir DF ..........164 2.6.6.9.16. Comparative Exposures and Safety Margins..........................................164 2.6.6.9.17. Conclusions 165

2.6.6.10. Tables and Figures ......................................................................................................167 2.6.6.11. References ...................................................................................................................168

LIST OF IN-TEXT TABLES

Table 1. Toxicology Program....................................................................................................17 Table 2. Exposure to TMC278 (Cmax and AUC) in an Oral Single-Dose Study with

TMC278 Base in Dogs ................................................................................................22 Table 3. Exposure to TMC278 (Cmax and AUC) in a 2-Week Oral Study with TMC278

in Mice.........................................................................................................................26 Table 4. Exposure to TMC278 (Cmax and AUC) in a 1-Month Oral Study with TMC278

in Mice.........................................................................................................................27 Table 5. Exposure to TMC278 (Cmax and AUC) in a 3-Month Oral Study with

TMC278 in Mice .........................................................................................................30 Table 6. Exposure to TMC278 (Cmax and AUC) in a 2-Week Oral Study with TMC278

Base in Rats .................................................................................................................34 Table 7. Exposure to TMC278 (Cmax and AUC) in a 2-Week Oral Study in Rats with

TMC278 Base and TMC278 .......................................................................................35 Table 8. Exposure to TMC278 (Cmax and AUC) in a 2-Week Oral Study in Rats with

TMC278 ......................................................................................................................36 Table 9. Exposure to TMC278 (Cmax and AUC) in a 1-Month Oral Bridging Study with

TMC278 Base and TMC278 in Rats ...........................................................................37 Table 10. Exposure to TMC278 (Cmax and AUC) in a 1-Month Toxicity Study of

TMC278 Base in Rats..................................................................................................38



Table 11. Exposure to TMC278 (Cmax and AUC) in a 6-Month Oral Toxicity Study with TMC278 Base in Rats..................................................................................................42

Table 12. Exposure to TMC278 (Cmax and AUC) in a 7-Day Oral Toxicity Study with TMC278 Base in Dogs ................................................................................................50

Table 13. Exposure to TMC278 (Cmax and AUC) in a 1-Month Oral Bridging Study with TMC278 Base and TMC278 in Dogs..........................................................................52

Table 14. Exposure to TMC278 (Cmax and AUC) in a 1-Month Toxicity Study Including 1-Month Recovery with TMC278 Base in Dogs .........................................................54

Table 15. Exposure to TMC278 (Cmax and AUC) in a 6-Month Toxicity Study with TMC278 Base in Dogs ................................................................................................57

Table 16. Exposure to TMC278 (Cmax and AUC) in a 12-Month Toxicity Study of TMC278 Base in Dogs ................................................................................................60

Table 17. Exposure to TMC278 (Cmax and AUC) in an 8-Week Oral Study with TMC278 in Juvenile Female Cynomolgus Monkeys...................................................................72

Table 18. Exposure to TMC278 (C1h and AUC) in a Single Oral Dose Bone Marrow Micronucleus Study with TMC278 Base in Mice........................................................80

Table 19. Mortality in a Mouse Carcinogenicity Study with TMC278........................................87 Table 20. Neoplastic Liver Changes in a Mouse Carcinogenicity Study with TMC278..............88 Table 21. Exposure to TMC278 (Cmax and AUC) in a Mouse Carcinogenicity Study with

TMC278 ......................................................................................................................89 Table 22. Mortality in the Rat Carcinogenicity Study with TMC278..........................................90 Table 23. Neoplastic Liver and Thyroid gland Changes in a Rat Carcinogenicity Study

with TMC278 ..............................................................................................................92 Table 24. Exposure to TMC278 (Cmax and AUC Values) in a Rat Carcinogenicity Study

with TMC278 ..............................................................................................................93 Table 25. Exposure to TMC278 (Cmax and AUC) in an Oral Embryo-Fetal Developmental

Study with TMC278 Base in Rats..............................................................................104 Table 26. Exposure to TMC278 (Cmax and AUC) in an Oral Embryo-Fetal Development

Study with TMC278 Base in Rabbits ........................................................................105 Table 27. Exposure to TMC278 (Cmax and AUC) in Rat Pups via Lactation (LD7) and

after 2 Weeks of Oral Dosing with TMC278.............................................................112 Table 28. Exposure of TMC278 (Cmax and AUC) in a Single Dose Oral Endocrinology

Study in Dogs with TMC278 Base ............................................................................125 Table 29. Qualification Summary for FTC Related Impurities and Degradation Products........142 Table 30. Qualified Levels of Residual Solvents and Proposed Limits for Emtricitabine .........143 Table 31. Qualified Levels for Impurities of Tenofovir DF.......................................................146 Table 32. ICH Classification of Organic Volatile Impurities for Tenofovir DF Drug

Substance ...................................................................................................................147 Table 33. Qualification of Impurities from TDF/FTC Tablets ..................................................149

LIST OF IN-TEXT FIGURES

Figure 1. Metabolites M30 (Left) and M31 (Right)....................................................................76 Figure 2. Structural Formulas of (Top) and (Bottom)................................136 Figure 3. Structural Formula ......................................................................................137 Figure 4. Structural Formulas of (Left) and (Right)...................................138 Figure 5. Synthetic Pathways for Adrenal Steroid Synthesis in Nonhuman Primates and

Man............................................................................................................................156

不純物B* 不純物C*

不純物D*不純物E* 不純物F*

*新薬承認情報提供時に置き換え



GLOSSARY OF ABBREVIATIONS AND DEFINITION OF TERMS ACTH adrenocorticotropic hormone A/G ratio albumin/globulin ratio ALP alkaline phosphatase ALT alanine aminotransferase APPT activated partial thromboplastin time AST aspartate aminotransferase BCOP bovine corneal opacity permeability BID twice daily BUN blood urea nitrogen CA citric acid CAC carcinogenicity assessment committee CASA computer-assisted sperm analysis CHO Chinese Hamster Ovary

,

CRF corticotrophin releasing factor CSF cerebrospinal fluid CYPxx cytochrome P450 isozyme xx DHEA dehydroepiandrosterone DMN dimethylnitrosamine DMSO dimethylsulfoxide DNA deoxyribonucleic acid DNCB 1-chloro-2,4-dinitrobenzene ECG electrocardiogram F female F1 first generation FDA United States Food and Drug Administration FDC fixed-dose combination FTC, TP-0006, GS-9019, 524W91

emtricitabine

GD gestation day GGT gamma-glutamyltransferase GI gastrointestinal GLP Good Laboratory Practices GS-4331-02 oral citric acid prodrug salt of tenofovir Hb hemoglobin HCl hydrochloride HCT hematocrit HIV-1 human immunodeficiency virus type 1 HPMC hydroxypropylmethylcellulose



GLOSSARY OF ABBREVIATIONS AND DEFINITION OF TERMS (CONTINUED) IC50 median inhibitory concentration ICH international conference on harmonization IP intraperitoneal LD lactation day LH luteinizing hormone M male MAA marketing authorization application MCH mean corpuscular hemoglobin MCHC mean corpuscular hemoglobin concentration MCV mean cell volume MPS mononuclear phagocytic system MTD maximum tolerated dose m/v mass per volume NCE normochromatic erythrocyte NK natural killer cells

NNRTI nonnucleoside reverse transcriptase inhibitor NOAEL no observed adverse effect level NOEL no observed effect level NRTI nucleoside/nucleotide reverse transcriptase inhibitor PBMC peripheral blood mononuclear cell PCE polychromatic erythrocyte PEG400 polyethylene glycol with molar mass between 380 and 420 PFC plaque forming cells PND postnatal day ppm parts per million = 10 6 pQCT peripheral quantitative computed tomography PR interval between the peak of the P wave and the peak of the R wave on ECG PT prothrombin time PTH parathyroid hormone QRS part of the ECG complex comprising the Q, R, and S-waves QT interval between the start of the Q wave and the end of the T wave on ECG QWBA quantitative whole body autoradiography RBC red blood cell, erythrocyte RDW red cell distribution width RPV, TMC278 rilpivirine (27.5 mg rilpivirine hydrochloride is equivalent to 25 mg rilpivirine) RR interval between the peak of R waves of 2 consecutive ECG complexes T3 triiodothyronine T4 tetraiodothyronine/thyroxine



GLOSSARY OF ABBREVIATIONS AND DEFINITION OF TERMS (CONTINUED) TDF, bis-POC PMPA Fumarate, GS-4331-05, PMPA Prodrug

tenofovir DF, Viread

TFV, GS-1278, PMPA

tenofovir

TK thymidine kinase TSH thyroid stimulating hormone TTC threshold of toxicological concern Tween 20 polyoxyethylenesorbitan monooleate 20 UDPGT T4-uridine diphosphate glucuronosyltransferase UDS unscheduled DNA synthesis US United States



PHARMACOKINETIC ABBREVIATIONS

z Terminal elimination rate constant, estimated by linear regression of the terminal elimination phase of the serum, plasma, or peripheral blood mononuclear cells (PBMCs) concentration of drug versus time curve

AUCinf The area under the serum, plasma, or PBMC concentration versus time curve extrapolated to infinite time, calculated as AUC0 last + (Clast/ z)

AUCx xx Partial area under the serum, plasma, or PBMC concentration versus time curve from time “x” to time “xx”

AUCtau The area under the serum, plasma, or PBMC concentration versus time curve over the dosing interval

C1h The concentration determined 1 hour after dosing. CL The systemic clearance of the drug after intravenous administration CL/F The apparent oral clearance after administration of the drug:

CL/F = Dose/AUCinf, where “Dose” is the dose of the drug Cmax The maximum observed concentration of drug in serum, plasma, or

PMBC T½ An estimate of the terminal elimination half-life of the drug in serum,

plasma, or PBMCs, calculated by dividing the natural log of 2 by the terminal elimination rate constant ( z)

Tmax The time (observed time point) of Cmax



2.6. NONCLINICAL SUMMARY

2.6.6. TOXICOLOGY WRITTEN SUMMARY

2.6.6.1. Brief Summary

This dossier is being submitted in support of a marketing authorization application (MAA) for a fixed-dose combination (FDC) film-coated tablet that contains the active substances emtricitabine (FTC), rilpivirine (RPV, which is also referred to as TMC278 throughout this document), and tenofovir disoproxil fumarate (tenofovir DF, TDF). Emtricitabine and TDF are antiretroviral agents developed by Gilead Sciences that have been approved for the treatment of human immunodeficiency virus type 1 (HIV-1) infection as stand-alone agents Emtriva® (Commission Decision granted on 20 [ ]) and Viread® (Commission Decision granted on 20 [ ]), and in a FDC product Truvada® (emtricitabine/tenofovir DF [FTC/TDF]; Commission Decision granted on 20 [ ]). Emtricitabine, a nucleoside reverse transcriptase inhibitor (NRTI), and TDF, a nucleotide reverse transcriptase inhibitor (NtRTI), are listed as preferred agents in United States (US) and international treatment guidelines {15207}, {12716}, {14065}. Emtricitabine and TDF are also approved for the treatment of HIV-1 infection in combination with efavirenz (EFV), a nonnucleoside reverse transcriptase inhibitor (NNRTI). This FDC product is Atripla (efavirenz/emtricitabine/tenofovir DF, [EFV/FTC/TDF]; Commission Decision granted on 20 [ ]). These products are currently approved in the US, the European Community, and other countries worldwide for use in adults. In some regions, Emtriva and Viread are approved for use in adolescents; Emtriva is also available as an oral solution and may be administered to children as young as 4 months of age. Rilpivirine, an NNRTI, is an investigational agent that is being submitted for approval by Tibotec BVBA.

Gilead Sciences has coformulated FTC and TDF, the standard of care NRTI backbone, with RPV into a FDC tablet. This FDC tablet is referred to as emtricitabine/rilpivirine/tenofovir disoproxil fumarate (FTC/RPV/TDF; dose strength 200/25/300 mg, respectively) throughout this document. Each FTC/RPV/TDF FDC tablet contains FTC, RPV, and TDF at the same dosages as recommended for the individual components, ie, 200 mg of FTC, 25 mg RPV (27.5 mg rilpivirine hydrochloride is equivalent to 25 mg RPV), and 300 mg of tenofovir disoproxil fumarate (equivalent to 245 mg tenofovir disoproxil or 136 mg of tenofovir [TFV]). The FTC/RPV/TDF FDC tablet has demonstrated bioequivalence to the individual dosage forms (FTC, TDF, and RPV). It is proposed that the FTC/RPV/TDF FDC tablet be indicated for the treatment of HIV-1 infection in adults and taken orally once daily with a meal.

In accordance with the advice received from the EMA (European Medicines Evaluation Agency) at the EMA Strategy meeting held on 20 (see meeting minutes provided in Module 1.2, Annex 5.14), the MAAs for RPV as a single agent and for the FTC/RPV/TDF FDC tablet are being submitted in parallel by Tibotec BVBA and Gilead Sciences, respectively.



Comprehensive programs of nonclinical studies with FTC, RPV, and TDF have been conducted. All of the definitive toxicology and toxicokinetic studies reported in this summary for FTC, RPV, TDF, and FTC/TDF were conducted in accordance with the Good Laboratory Practice (GLP) procedures promulgated by international health authorities. Pilot, exploratory, and mechanistic studies were not all conducted under strict GLP procedures but were conducted using appropriate protocols and documentation to assure data integrity. Within this Module 2.6.6 and in the toxicology Tabulated Summary 2.6.7, RPV is referred to as TMC278 in the text and tables describing the nonclinical studies of rilpivirine alone. In summary sections, and in the Discussion and Conclusions (Section 2.6.6.9), RPV is used preferentially.

As the RPV component is a new chemical entity, this FDC MAA dossier contains full data on this new component while providing the key data on the Truvada (i.e., FTC, TDF, and FTC/TDF) components. All FTC, TDF, and FTC/TDF studies considered to support the FDC are included to ensure that this is a ‘stand-alone dossier.’ This is in agreement with the feedback received at the presubmission meeting with the EMA on 20 and with the meeting with the Rapporteur/Co-Rapporteur on 20 (see Module 1.2.5.14, final minutes). To assist the reviewer, a listing of all the FTC, TDF, FTC/TDF, and EFV/FTC/TDF nonclinical reports is provided in Module 2.4, Section 2.4.7. Nonclinical study reports are provided in Module 4.

Information from all nonclinical studies with FTC, RPV, and TDF should be considered in the context of the substantial clinical experience with FTC and TDF within antiretroviral combination therapy for the treatment of HIV-1 infection, the Phase 2 and Phase 3 clinical experience with RPV, and with RPV administered in combination with Truvada (FTC/TDF).

This nonclinical summary provides an overview of nonclinical information that is relevant to the assessment of the FDC of FTC, RPV, and TDF.

The nonclinical data discussed within this document demonstrate an acceptable benefit/risk profile for the proposed use of FTC/RPV/TDF FDC tablets for the treatment of HIV-1 infection in adults. All information from nonclinical studies that is of relevance to the prescriber and patient has been included in the proposed Prescribing Information and Patient Leaflet.

Emtricitabine

The general systemic (single and repeat dose) toxicity, reproductive toxicity, and mutagenicity of FTC have been characterized in a variety of in vitro and in vivo studies.

The preclinical toxicity studies demonstrated that FTC is generally well tolerated for up to a year at doses producing systemic exposure levels in animals much greater than those anticipated in patients treated with the recommended clinical dose.



Treatment-related effects were confined to high-dose groups only and included changes in red blood cell (RBC) parameters, interpreted as a mild, reversible anemia (mice 1 month, 6 months; rat 3 months; monkey 1 year); changes in various organ weights without any associated adverse histopathological effects in rodents (mouse 1 month, 6 months; rat 3 months), increased urine output (mice 6 months) and soft feces (monkeys 1 month, 3 months). No observed effect levels (NOELs) could be established for all treatment-related effects, and in several cases the minor effects observed were reversible after a recovery period.

Emtricitabine had no unwanted pharmacologic activity as determined in a variety of in vitro and in vivo pharmacology studies, including no pharmacological effects on the cardiovascular system.

Emtricitabine was not genotoxic or clastogenic, and did not adversely affect reproduction or embryo-fetal development. It was relatively non-cytotoxic to human cells in vitro, including bone marrow progenitor cells, and did not produce mitochondrial toxicity.

Emtricitabine long-term carcinogenicity studies in mice and rats showed no evidence of carcinogenic potential at exposures greater than 25 times those observed in humans at the therapeutic dose.

Rilpivirine

TMC278 is a next generation NNRTI active against wild type and NNRTI-resistant HIV-1.

The following convention is applied throughout this module: reference is made to “TMC278” when the hydrochloride (HCl) salt was administered and to “TMC278 base” when the base was administered. The dose or concentration is always given as base equivalent. The analyte in bioanalytical determinations is referred to as “TMC278.”

Rilpivirine base has been applied in the early phases of development and TMC278 in the later phases, after selection of the final chemical form.

The objectives of the toxicology program were to evaluate the toxicological profile of TMC278, its metabolites, and impurities in nonclinical test systems. Furthermore, attempts were made to elucidate the mechanism of action of the effects observed. Toxicological profiles and mechanisms of action were used in the assessment of a safety margin (presented in Module 2.4, Nonclinical Overview) between the dose and exposure needed for an efficacious and safe treatment of HIV-1 infection, and the adverse effects induced in the nonclinical test systems.



All in vivo studies were done by oral administration, with the exception of the sensitization and dermal irritation studies. For oral dosing, TMC278 base was dissolved in polyethylene glycol 400 (PEG400) usually with 100 mg/mL citric acid (CA) to improve exposure, with the exception of the rabbit studies in which TMC278 base was suspended in 0.5% (m/v; mass per volume) aqueous hydroxypropylmethylcellulose (HPMC). TMC278 was suspended in 0.5% (m/v) aqueous HPMC in the mouse, rat, and dog studies. In the studies with cynomolgus monkeys, the vehicle was 1% (m/v) aqueous HPMC with 0.5% Tween 20.

No formal single-dose studies were done as single dose evaluations were part of the initial dose range finding studies or, in the case of mice, part of the bone marrow micronucleus test. The single-dose toxicity of TMC278 in mice, rats, and dogs appeared to be low. The maximal feasible dose did not induce significant toxicity or effects.

Repeat-dose toxicity studies were done in mice as preparation for a 3-month dose range finding carcinogenicity study; in rats for up to 6 months; in nonpregnant rabbits for 5 days as preparation for the dose range finding early embryonic development studies; in dogs up to 12 months; and in immature female cynomolgus monkeys up to 8 weeks as part of the assessment of the effects of TMC278 on juvenile animals. Juvenile rats were used in a 2-week oral dosing study starting on lactation Day 12 (LD 12). The dogs in studies up to 1-month duration are also considered immature. Since the animals were 6.5 to 8 months old at the start of these studies they were not yet sexually mature at the end of the dosing period. Reversibility (after a 1-month recovery period) of the effects of TMC278 on the high dose group was investigated in the 6-month rat study and the 1-month dog study.

The targets of toxicity of TMC278 identified in the repeat-dose studies were: RBCs (mouse, rat, and dog); coagulation (rat); liver (rat and dog); kidneys (mouse and dog); thyroid gland with secondary effects on the pituitary gland (rat); adrenal glands (mouse, rat, dog, and cynomolgus monkey); testes (dog); and ovaries (dog, in immature females with secondary effects on other tissues of the genital tract and on mammary glands). The majority of the induced effects appeared to be completely reversible after a 1-month postdosing period. The effects on thyroid gland and coagulation in rats and on liver and serum alkaline phosphatase (ALP) in dogs showed signs of recovery, but this was not complete at the end of the 1-month postdosing period. A number of targets were affected at the low dose tested in dogs and cynomolgus monkeys preventing establishment of no observed adverse effect levels (NOAELs) in these 2 species.

TMC278 did not show a potential for genotoxicity in the in vitro bacterial reverse mutation (Ames) tests and mouse lymphoma assays and the in vivo mouse bone marrow micronucleus test. No potential for carcinogenicity by a direct interaction with deoxyribonucleic acid (DNA) was concluded in the 2-year studies in mice and rats. The hepatocellular adenomas and carcinomas seen in mice are considered to be induced by liver enzyme induction, an epigenetic mechanism. The hepatocellular adenomas and thyroid follicular cell adenomas and carcinomas in rats are also considered to derive from an epigenetic mechanism as result of liver enzyme induction and a likely associated increased clearance of thyroid hormones leading to a continuous stimulation of the thyroid gland by thyroid stimulating hormone (TSH). No neoplastic lesions associated with the other targets of TMC278 were detected.



TMC278 did not show a teratogenic potential and did not affect fertility, fecundity, early embryonic development, maternal behavior at parturition and during lactation, or peri- and postnatal development of offspring from dams treated with TMC278.

TMC278 had similar targets or induced the same type of adverse effects in juvenile or sexually immature rats, dogs, and cynomolgus monkeys as in adult animals. The TMC278-related effects on the female genital tract and mammary glands in dogs at the end of the 1-month studies differed from the effects in longer duration studies in which animals were sexually mature animals at the end of the dosing period. The activation of ovaries in dogs treated with TMC278 in the 1-month studies led to secondary activation noticed in the other parts of the genital tract and mammary glands. These secondary effects occur normally in sexually mature animals during estrous cycle. Therefore, they were not noted as a difference between TMC278-treated and control animals in longer duration studies. However, in the 1-month study, the control animals were still dormant (i.e., no activation of ovaries and therefore no secondary effects). In the evaluation relative to control animals, the secondary effects were noted. Activation of ovaries was not induced during an 8-week study in immature cynomolgus monkeys at the age of approximately 18 months at the start of the study.

TMC278 tested negative for the potential to cause phototoxicity, skin irritation, and delayed-type hypersensitivity and to induce an immunotoxic effect on the challenge of rats with sheep RBCs. TMC278 was classified as a moderate eye irritant in an in vitro test.

The drug substance contains 3 impurities that need to be qualified according to Guideline Q3A of the International Conference on Harmonization (ICH), entitled: “Impurities in New Drug Substances.” Two of these, and , have been evaluated upon spiking to the drug substance at a level of 4%. Qualification comprised a bacterial reverse mutation Ames test, a mouse lymphoma assay, and a 1-month oral rat study. The presence of the impurities at 4% did not modify the effects of TMC278 in any of the tests. The third impurity, , is the -isomer of TMC278. This isomer was present in all drug substance batches involved in pivotal nonclinical studies at the level of minimally 0.61%. In view of the close structural relationship with TMC278 and the overage between the lowest nonclinical dose (5 mg/kg/day) and the recommended clinical dose of 25 mg once daily, separate qualification of is not considered relevant.

Three further (potential) impurities, , , and , which contain moieties with a mutagenic alert, are present in the drug substance at levels that do not warrant qualification according to ICH Q3A. The mutagenic potentials of the HCl salts of ,

, and of and were tested in an Ames test. Only showed a mutagenic potential. So it is concluded that is a genotoxic impurity. The maximum allowable level of in a daily dose of 25 mg TMC278 was calculated to be

ppm, on the basis of the Threshold of Toxicological Concern (TTC) approach with daily treatment for longer than 12 months (Committee for medicinal product for human use [CHMP]. Guideline on the limits of genotoxic impurities. CPMP/SWP/5199/02. EMEA/CHMP/QWP/251344/2006 28 June 2006). The level of in drug substance and drug product has been controlled at less than ppm.


不純物A*

不純物D* 不純物E* 不純物F*

不純物A*

不純物D*

不純物D*

不純物D’*

不純物D*

不純物D*

不純物D’* 不純物E* 不純物F*




Tenofovir Disoproxil Fumarate

The toxicologic evaluation of TDF administered by oral gavage included acute toxicity studies in rats and dogs; subchronic toxicity studies in mice, rats, woodchucks, dogs, and monkeys; chronic toxicity studies in rats and dogs; genotoxicity and carcinogenicity studies; reproductive toxicity studies in rats and rabbits; and local tolerance and antigenicity studies in rabbits and guinea pigs. These studies have enabled the toxicologic profile of TDF to be well characterized in 3 animal species.

The principal target organs of toxicity following oral administration of TDF were the kidney, bone, and gastrointestinal (GI) tract (in rodents). Based upon the nonclinical findings, potential clinical adverse events should be easily detected and reversible upon discontinuation of treatment. More details of the principal toxicity studies are presented in the tabulations (Module 2.6.7) and a summary of the principal toxicologic findings are discussed below.

The dose-limiting toxicity in rodents was GI. This effect was related, not to systemic exposure, but to local gut exposure that, on a direct dosage (mg/kg/day) basis, was 6- to 20-fold higher than in humans. The dose-limiting toxicity associated with TDF administration in dogs and monkeys was nephrotoxicity. Dose-related histopathological changes included tubular dilation, degeneration/regeneration, and/or interstitial nephritis. Karyomegaly in the renal tubular epithelium, reported at relatively low multiples of the human dose of all species, was considered a morphologic change without pathologic consequence. Reductions of serum phosphate were observed in rats and monkeys. Oral phosphate supplementation was shown to normalize the serum phosphate levels in rats and monkeys. Chronic rat and dog studies have shown some loss of bone mineral density at relatively high doses that appears to be nonprogressive or minimally progressive between Weeks 13 and 42, with incomplete resolution after a 13-week recovery period. The osteopenia may be related to accelerated bone resorption secondary to reduction of the intestinal phosphate absorption and/or a mild renal phosphate leak that develops with chronic treatment. Clinically relevant bone pathology (osteomalacia) has been documented only in rhesus monkeys in which tenofovir (TFV, GS-1278, PMPA) was chronically administered by daily subcutaneous injection to infant and juvenile monkeys at AUCss levels that were more than 30-fold those of humans at the 300-mg/day dose.

In the 13- and 42-week rat and dog studies and the 56-day monkey study, there was a mild rise in serum alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) activities and, in the chronic rat and dog studies, there was a slight elevation in the serum bilirubin concentration; there was no concurrent microscopic alterations in the liver of the rat and monkey and only slight centrilobular congestion and pigment accumulation in the dog. The biochemical alterations resolved at the end of a 13-week recovery period in the rat and dog.



There were no adverse effects on fertility or embryo-fetal development in rats or rabbits. A developmental and perinatal/postnatal reproduction toxicity study evaluated the adverse effects of TDF treatment on gestation, parturition, lactation, and maternal behavior in female Sprague-Dawley rats from implantation through lactation and weaning, and on the development of the offspring from the treated female rats. No adverse effects on mating performance or reproductive parameters were noted. Increased peri/postpartum pup mortality, reduced pup survival, and reduced pup body weights were observed at maternally toxic doses. The first generation (F1) male and female NOEL for behavioral, reproductive, and developmental toxicity of TDF was 150 mg/kg/day (exposure 4 the human dose). Sexual maturation was affected when doses of 450 and/or 600 mg/kg/day were administered.

Tenofovir DF was not mutagenic in the in vitro Salmonella/Escherichia coli mammalian microsome reverse assay with the exception of an equivocal response in 1 of 3 tests in Salmonella strain TA1535 without metabolic activation. Tenofovir DF was positive for inducing forward mutations in the in vitro L5178Y mouse lymphoma cell assay in the presence or absence of S-9 metabolic activation and was weakly positive in a study to detect DNA damage by measuring unscheduled DNA synthesis (UDS) induced in vivo in rat primary hepatocytes cultured in vitro. However, TDF was negative in an in vivo mouse micronucleus assay.

The rat carcinogenicity study showed no evidence of oncogenic potential at doses providing up to 5 times the human systemic exposure. A low incidence of duodenal tumors observed at the highest dose in the mouse carcinogenicity study is thought to be due to local effects.

Emtricitabine/Tenofovir DF

Emtricitabine and TDF have been assessed individually in comprehensive regimens of toxicity studies that demonstrate their suitability for use as indicated. Due to the lack of pharmacokinetic interaction and low potential for toxicologic interaction based on non-overlapping target organs of toxicity, no potentiation of toxicity was expected with the combination of FTC and TDF. Several short-term studies were conducted to confirm this supposition.

Daily oral administration of non-degraded and degraded FTC/TDF for 14 consecutive days to male Sprague-Dawley rats was well tolerated. There were no toxicologically significant differences between non-degraded and degraded FTC/TDF tablets or any evidence of new or exacerbated toxicities as compared to similar duration studies in rats with the agents administered alone.

Repeat dose toxicity of the combination of FTC/TDF in dogs treated for 1 month was similar to that of the agents alone, indicating that combining these agents did not alter or exacerbate target organ toxicity.

The combination of FTC/TDF was negative in the Ames assay and positive in the Mouse Lymphoma assay. There was no evidence of exacerbation of mutagenic potential with the combination as compared to the agents alone.



The impurities and degradation products in the 2 active ingredients, FTC and TDF, have been identified and qualified in toxicology studies.

Emtricitabine/Rilpivirine/Tenofovir DF

In line with feedback from EMEA (EMEA/CHMP/SAWP/670243/2009 corrigendum, Module 1.2.5.14), no new nonclinical safety studies with the combination of the 3 agents were considered necessary given the nonoverlapping toxicity profiles of the individual compounds.

Further, based on the well-characterized toxicity profiles of FTC, RPV, and TDF; the clinical safety data available for the approved agents FTC, TDF, the FTC/TDF FDC product Truvada; and the clinical data from Phase 2, and Phase 3 studies conducted with Truvada and RPV administered in combination, the data demonstrate an acceptable benefit/risk profile for the proposed use of the FDC product for the treatment of HIV-1 infection in adults.



Table 1. Toxicology Program

Study Type & Duration Route of

Administration Species Compound

Administered Single Dose Toxicity po, iv Mouse, rat FTC po Mouse, rat, dog RPV base po Rat, dog TDF Repeat Dose Toxicity FTC 2 Weeks po Mouse FTC 1 Month po Mouse, monkey FTC 3 Months po Rat FTC 6 Months po Mouse FTC 1 Year po Monkey FTC RPV 5 Days po Rat, rabbit, dog RPV base 7 Days po Dog RPV base 2 Weeks po Mouse, rat RPV base (rat),

RPV (mouse, rat) 1 Month po Mouse, rat, dog RPV base (rat,

dog), RPV (mouse, rat, dog)

8 Weeks po Monkey RPV 3 Months po Mouse RPV 6 Months po Dog, rat RPV base 1 Year po Dog RPV base TDF 5 Days po Rat, dog Tenofovir citric

acid salt prodrug 13 Days po Mouse TDF 14 Days po Rat TDF 28 Days po Rat, dog TDF 13 Weeks po Mouse, rat & dog TDF 42 Weeks po Rat, dog TDF FTC/TDF 14 Days po Rat TDF/FTC 28 Days po Dog TDF/FTC Genotoxicity FTC In Vitro: Mutation and Chromosomal

Aberration Assays Bacteria, mouse,

hamster FTC

In Vivo: Micronucleus Assay po Mouse FTC RPV In Vitro: Nonmammalian Cell System Bacteria RPV base, RPV In Vitro: Mammalian Cell System Mouse RPV base, RPV In Vivo: Micronucleus Assay po Mouse RPV base TDF In Vitro: Mutation Assays Bacteria, mouse,

rat TDF/tenofovir

In Vivo: Micronucleus Assay po Mouse TDF





Administered In Vivo: Unscheduled DNA Synthesis

Test po Rat TDF

FTC/TDF In Vitro: Mutation Assays Bacteria, mouse TDF/FTC Carcinogenicity FTC 2 Years po Mouse, rat FTC RPV 2 Years po Mouse, rat RPV TDF 2 Years po Mouse, rat TDF Reproduction Toxicity FTC Fertility & Early Embryonic

Development po Mouse, rat FTC

Embryo-Fetal Development po Mouse, rabbit FTC Prenatal & Postnatal Development po Mouse FTC RPV Fertility & Early Embryonic

Development po Rat RPV

Embryo-Fetal Development po Rat, rabbit RPV base Prenatal & Postnatal Development po Rat RPV TDF Fertility & Early Embryonic

Development po Rat TDF

Embryo-Fetal Development po Rat, rabbit TDF Prenatal & Postnatal Development po Rat TDF Local Tolerance Phototoxicity - In Vitro: Mammalian Cell

System Mouse RPV base

Eye, Skin Irritation Tests Topical Bovine, rabbit RPV Eye, Skin Irritation Tests Topical Rabbit TDF Other Studies FTC Immunotoxicity po Rat FTC Oral Qualification Studies po Mouse FTC Genotoxicity Qualification Assays in vitro – FTC RPV Antigenicity Topical Mouse RPV Mechanistic Studies – In Vitro – RPV Mechanistic Studies – In Vivo po Dog RPV base Impurities - Genotoxicity Mutation

Assays in vitro – RPV/ /

/ /

Oral Qualification Studies po Rat RPV/ ,

TDF Antigenicity Topical Guinea pig TDF Mitochondrial Toxicity in vitro – Tenofovir

不純物B*

不純物E*

不純物F*

不純物C*

不純物B*不純物C*






Administered Mitochondrial Toxicity po Woodchuck TDF Mitochondrial Toxicity/Interaction Study po Rat TDF & Adefovir

Dipivoxil Toxicity to Bone, Effects on

Phosphate/Phosphorus & Ca Concentrations

in vitro – Tenofovir

Toxicity to Bone, Effects on Phosphate/Phosphorus & Ca Concentrations

po (sc) Rat, monkey TDF (tenofovir)

Impurities po Rat TDF/degraded TDF

Genotoxicity Mutation Assays in vitro – Tenofovir Safety Studies in Efficacy Models sc Monkey Tenofovir RPV = TMC278; RPV base = TMC278 base

2.6.6.2. Single-Dose Toxicity

2.6.6.2.1. Emtricitabine: Mouse

FTC: An Acute Oral Toxicity Study in the Mouse with 524W91 (TTEP/93/0020)

Five male and 5 female CD-1 mice were orally administered 4000-mg/kg FTC (Tabulated Summary 2.6.7.5.A, TTEP/93/0020). The animals were observed daily for clinical symptoms of toxicity and weighed on Days 0, 7, 13, and 14. On Day 14, they were necropsied. There were no deaths or clinical findings. Emtricitabine was well tolerated and the NOEL was 4000 mg/kg.

FTC: An Acute Intravenous Toxicity Study in the Mouse with 524W91 (TTEP/93/0023)

Five male and 5 female CD-1 mice were intravenously administered 200 mg/kg FTC (Tabulated Summary 2.6.7.5.A, TTEP/93/0023). The animals were observed daily for clinical symptoms of toxicity and weighed on Days 0, 7, 13, and 14. On Day 14, they were necropsied. There were no deaths or clinical findings. Emtricitabine was well tolerated and the NOEL was 200 mg/kg.

2.6.6.2.2. Rilpivirine: Mouse

In a bone marrow micronucleus study (Tabulated Summary 2.6.7.9.B, TMC278-Exp5538 and Addendum [FK4259]), a single oral dose of TMC278 base dissolved in PEG400 with CA was administered by gavage to 10 male and 10 female CD-1 mice per group. The doses were 0 (vehicle), 100, 400, and 1600 mg/kg given in a volume of 20 mL/kg. The dose of 1600 mg/kg was the maximally feasible dose on the basis of the maximally feasible volume for a single dose study, 20 mL/kg, and a supersaturated and highly viscous solution of 80 mg/mL. A limited number of samples for confirmation of exposure were taken from the animals over a period of 6 hours after dosing. Five animals of each sex per group were killed 24 and 48 hours after dosing.



TMC278 did not cause any mortality. There were no abnormalities in clinical observations or relevant effects on body weight. At necropsy, no TMC278-related gross lesions were recorded. At 1600 mg/kg, mean plasma concentration determined 1 hour after dosing (C1h) was 60 and 58 μg/mL and the AUC0 6h was 307 and 287 μg.h/mL in males and females, respectively. These values were essentially similar to those at 400 mg/kg, indicating saturation of absorption (Tabulated Summary 2.6.7.9, TMC278-Exp5538).

2.6.6.2.3. Emtricitabine: Rat

FTC: An Acute Oral Toxicity Study in the Rat with 524W91 (TTEP/93/0021)

Five male and 5 female CD rats were orally administered 4000-mg/kg FTC (Tabulated Summary 2.6.7.5.A, TTEP/93/0021). The animals were observed daily for clinical symptoms of toxicity and weighed on Days 0, 7, 13, and 14. On Day 14, they were necropsied. There were no deaths or clinical findings. Emtricitabine was well tolerated and the NOEL was 4000 mg/kg.

FTC: An Acute Intravenous Toxicity Study in the Rat with 524W91 (TTEP/93/0024)

Five male and 5 female CD rats were intravenously administered 200 mg/kg FTC dissolved in 0.9% saline (Tabulated Summary 2.6.7.5.A, TTEP/93/0024). The animals were observed daily for clinical symptoms of toxicity and weighed on Days 0, 7, 13, and 14. On Day 14, they were necropsied. There were no deaths or clinical findings and FTC was well tolerated. It was concluded that the approximate lethal dose in rats dosed intravenously was 200 mg/kg.

2.6.6.2.4. Rilpivirine: Rat

The highest feasible concentration of an oversaturated solution of TMC278 base in PEG400 was 80 mg/mL, in terms of viscosity for administration by gavage to rats. This concentration was administered by oral gavage at a volume of 10 mL/kg to 6 male and 6 female Sprague Dawley rats, rendering a dose of 800 mg/kg (Tabulated Summary 2.6.7.5.B, TMC278-Exp5559; Tabulated Summary 2.6.7.2.B, FK4278 [Exp5559]). Mortality and clinical observations were evaluated for 1 day following the administration. Four males and 4 females were sampled to assess exposure.

TMC278 did not cause any mortality. All males and 2 out of 6 females showed salivation during the day of dosing. At necropsy, no TMC278-related gross lesions were recorded. The mean Cmax was 8.0 and 18.0 μg/mL and the AUCinf was 86 and 233 μg.h/mL in males and females, respectively.



2.6.6.2.5. Tenofovir DF: Rat

An Acute Oral Gavage Toxicity Study of Tenofovir DF (GS-4331-05) in the Albino Rat Followed by a 14-Day Observation Period (R990200)

The objective of this study was to investigate the potential acute toxicity of TDF at doses of 0, 160, 500, or 1500 mg/kg following a single oral gavage administration to the rat (5/sex/group) followed by a 14-day observation period (Tabulated Summary 2.6.7.5.C,R990200). Clinical observations (twice daily [BID]), body weights (weekly), and food consumption (weekly) were determined; hematology, clinical biochemistry, and urinalysis samples were taken at the time of necropsy for evaluation; macroscopic observations were noted at necropsy; and histopathology was performed on selected tissues.

There were no deaths or treatment-related clinical signs or effects on body weight or food consumption. Hematology, clinical biochemistry, and urinalysis parameters were considered unaffected by treatment. There were no treatment-related macroscopic or microscopic findings. In conclusion, a single oral gavage administration of TDF followed by a 14-day observation period at dose levels of 160, 500, and 1500 mg/kg did not produce any evidence of an adverse reaction to treatment. The NOAEL is therefore considered to be 1500 mg/kg.

2.6.6.2.6. Rilpivirine: Dog

For dogs, the highest feasible concentration of an oversaturated solution of TMC278 base in PEG400 with or without CA was 100 mg/mL in terms of viscosity for the administration by gavage. The dogs were pretreated with the vehicles applied in this study. Polyethylene glycol 400 was administered by oral gavage at 0.8 mL/kg to 2 groups of 1 male and 1 female beagle dog and PEG400 + CA to another group (Tabulated Summary 2.6.7.5.B, TMC278-Exp5461; Tabulated Summary 2.6.7.2.B, TMC278-Exp5461 [FK4102]). The next day, the PEG400-pretreated animals were dosed with TMC278 base dissolved in PEG400 at a single dose of 40 or 80 mg/kg. The PEG400 + CA pretreated animals received TMC278 base dissolved in PEG400 + CA at a single dose of 80 mg/kg. The animals were observed for 4 days after dosing. No clinical pathology or necropsy was performed on these animals.

TMC278 did not cause any mortality. Only in the group dosed with 80 mg/kg dissolved in PEG400 did a higher incidence of vomiting occur for 2 days after dosing. Slightly soft feces were recorded for the male at Day 3 after dosing and for the female at Days 2 and 3 after dosing. The animals dosed with 40 mg/kg were transferred to the repeat-dose arm of the study 3 weeks after the end of the single dose observation period. Exposure data are presented in Table 2 (Tabulated Summary 2.6.7.5B, TMC278-Exp5461).



Table 2. Exposure to TMC278 (Cmax and AUC) in an Oral Single-Dose Study with TMC278 Base in Dogs

Cmax (μg/mL) AUC0 24h (μg.h/mL) Dose(mg/kg/day) Vehicle M (n = 1) F (n = 1) M (n = 1) F (n = 1)

40 PEG400 1.1 0.95 21 19

80 PEG400 0.88 0.81 17 16

80 PEG400 + CA 2.1 2.2 42 49

PEG = polyethylene glycol, CA = citric acid, M = males, F = females

2.6.6.2.7. Tenofovir DF: Dog

An Acute Oral Gavage Toxicity Study of Tenofovir DF (GS-4331-05) in the Beagle Dog Followed by a 14-Day Observation Period (D990201)

The objective of this study was to investigate the potential acute toxicity of TDF at doses of 0, 30, 90, or 270 mg/kg following a single oral gavage administration to the dog (1/sex/group) followed by a 14-day observation period (Tabulated Summary 2.6.7.5.C,D990201). Clinical observations (BID), body weights (weekly), and food consumption (weekly) were determined. Hematology and clinical biochemistry samples were collected on Days 2 and 14 for evaluation and urinalysis samples were collected on Days 3 and 12. Macroscopic observations were noted at necropsy and histopathology was performed on selected tissues.

There were no deaths. Emesis and ptylism were observed 1 hour postdose in the 270-mg/kg group and in the male control dog and resolved 2 hours postdose. There were no effects on body weight or food consumption. Hematology, clinical biochemistry, and urinalysis parameters were considered unaffected by treatment. There were no treatment-related macroscopic findings. Microscopic alterations included renal cortical tubular basophilia at dosages of 90 and 270 mg/kg and minimal renal tubular epithelial karyomegaly at 270 mg/kg.

In conclusion, a single oral gavage administration of TDF at doses of 30, 90, and 270 mg/kg to the beagle dog followed by a 14-day observation period resulted in TDF-related minimal renal tubular epithelial karyomegaly (270 mg/kg) and renal tubular basophilia (90 and 270 mg/kg). The NOEL for TDF is considered to be 30 mg/kg.

2.6.6.2.8. Emtricitabine/Rilpivirine/Tenofovir DF

No single-dose studies have been performed with the combination of FTC, RPV, and TDF. Single dose toxicity studies indicated that all 3 agents had low acute toxicity. With no overlapping toxicities, coadministration is unlikely to change the acute toxicity profile.



2.6.6.3. Repeat-Dose Toxicity

2.6.6.3.1. Mouse

2.6.6.3.1.1. Emtricitabine: 14-Day Oral (Gavage) Toxicity Study in Mice Given FTC (IUW00701)

Four groups of 15 male and 15 female CD-1 mice were given FTC, suspended in 0.5% methyl cellulose, at doses of 0, 120, 600, and 3000 mg/kg/day (Tabulated Summary 2.6.7.7.A, IUW00701). The dose was given by gavage and as a divided dose with 6 hours between the morning and afternoon dose. Ten mice/sex/group were necropsied at the end of the exposure period; 5/sex/group were necropsied at the end of a 2-week postdose recovery period. There were no important differences in levels of FTC measured when males were compared to females and Day 2 values were compared to Day 14 values. Mean concentrations for FTC were proportional to dose. At the high dose, mean concentrations on Day 2 were 318 and 358 g/mL for males and females, respectively; and on Day 14 were 389 and 405 g/mL, respectively. There were no signs of toxicity.

2.6.6.3.1.2. Emtricitabine: A 30 Day Oral Toxicity Study in Mice Given 524W91 (TOX599)

Groups of 14 male and female mice were orally administered (once daily) FTC suspended in 0.5% methylcellulose at 0, 120, 600, and 3000 mg/kg/day for 30 days, with 4 males and females from each group held for an additional 13-day recovery period (Tabulated Summary 2.6.7.7.B, TOX599). Also exposed to the same dosing regimen were groups of 10 males and females, which were used for clinical pathology evaluations prestudy and on Days 31 to 32. Groups of 38 male and female satellite toxicokinetic animals were dosed at 120, 600, and 3000 mg/kg/day and drug level determinations made from samples collected on Days 3 and 32. There were no clinical signs of toxicity that were thought to be treatment related. Two high dose male mice died of undetermined causes, and a relationship to treatment cannot be ruled out. No treatment-related changes in feed consumption or body weight were seen. At the end of the study, hemoglobin (Hb) and hematocrit (HCT) were reduced in high-dose females, and total erythrocyte count (RBC) was decreased in both sexes, whereas mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW) were increased in both sexes. All the hematology changes were reversed in females following the recovery period, but high-dose males experienced a further decline in HCT and RBC, with some improvement in MCV and RDW. There were no changes in clinical chemistry values, urinalysis parameters, or ocular toxicity endpoints. Pituitary, heart, spleen, and ovary weights were increased in high-dose females and the spleen weight was increased in high-dose males, whereas the testes and thymus weights were decreased. There were no accompanying histopathologic correlates noted. A NOEL for hematological changes was established at 600 mg/kg/day. Day 32 toxicokinetic AUCinf values for high dose males and females were 7830 and 6130 M·h (1680 and 1320 g·h/mL), respectively. Day 3 values were similar.



2.6.6.3.1.3. Emtricitabine: 6-Month Oral (Gavage) Toxicity Study in Mice Given FTC with 3-Month Interim Kill (TOX022)

Groups of 30 male and 30 female CD-1 mice were administered FTC at 0, 167, 500 and 1500 mg/kg/day, given as a single oral dose each day for 26 weeks (Tabulated Summary 2.6.7.7.C, TOX022). After 13 weeks of treatment, 10 males and 10 females from each group were killed and necropsied. After 26 weeks of treatment, 15 males and 15 females from each group were killed and necropsied. The remaining 5 males and 5 females from each group went untreated for 1 month and were then necropsied. Satellite groups of 40 males and 40 females per dose group were used for toxicokinetic blood collections after 13 and 26 weeks. Clinical observations were made daily and ophthalmoscopic exams at predose and Weeks 13 and 26. Unfasted blood samples were taken at each necropsy for clinical pathology evaluations. At necropsy, organs were examined grossly, weighed, and processed for histopathologic evaluations.

Compared to controls, there were no significant clinical observations, body weight changes, ophthalmoscopic findings, blood chemistry alterations, organ weight changes, or gross and histopathologic findings. Mean cell volume (MCV) and MCH were elevated in the high-dose group after 26 weeks of exposure, more so in females than males. These changes were still evident after the 1-month recovery period.

At 3 and 6 months of exposure, plasma samples were collected from 3 satellite animals/sex/group at predose, 0.5, 1, 2, 5, and 8 hours after the daily dose. With the exception of females in the low dose group, mice at all doses had measurable concentrations of FTC in the predose blood samples. Peak plasma concentrations (Cmax) of FTC were reached by 30 to 60 minutes after dosing. Following 3 months exposure, Cmax values at 167 mg/kg/day were 44.0 and 37.2 g/mL; at 500 mg/kg/day were 128.0 and 88.8 g/mL; and at 1500 mg/kg/day were 225.0 and 187.1 g/mL for males and females, respectively. Following 6 months exposure, Cmax values at 167 mg/kg/day were 41.7 and 48.6 g/mL; at 500 mg/kg/day were 87.3 and 111.7 g/mL; and at 1500 mg/kg/day were 310.9 and 306.0 g/mL for males and females, respectively.

The systemic exposure to FTC over the steady state dosing interval (AUC0 24h) increased proportionally as the dose increased. Following 3 months exposure, AUC0 24h increased from 63.8 and 67.9 g·h/mL at 167 mg/kg/day to 513.4 and 569.5 g·h/mL at 1500 mg/kg/day for males and females, respectively. Following 6 months exposure, AUC0 24h increased from 82.3 and 93.1 g·h/mL at 167 mg/kg/day to 732.1 and 899.4 g·h/mL at 1500 mg/kg/day for males and females, respectively. A NOEL of 500 mg/kg/day was established (equivalent to an AUC0 24h of 266.4 g·h/mL).



2.6.6.3.1.4. Emtricitabine: A 6 Month Oral Toxicity Study (with a 3 Month Interim Sacrifice) in Mice Given 524W91 (TOX628)

Groups of 25 male and 25 female CD-1 mice were given once daily doses of FTC at 0, 120, 600, or 3000 mg/kg for up to 6 months (Tabulated Summary 2.6.7.7.D, TOX628). Five mice/sex/dose were subjected to a 3-week recovery period following 6-months of dosing. An additional 28/sex/group were used to assess plasma concentrations of drug, and 10/sex/dose (20 in the control group) were used for clinical pathology evaluations. The following parameters were evaluated for evidence of toxicity: body weights, feed consumption, clinical signs, hematology, clinical chemistry, urinalysis, ophthalmoscopic examinations, organ weights, gross and histopathology, and electron microscopy.

After 6-months of exposure at the high dose, there were reversible decreases in RBCs, and reversible increases in MCV and urine quantity for both sexes. In high dose females, there was a reversible increase in absolute and relative thyroid weight, which was not associated with histopathological changes. The AUC0 24h for sexes combined on Day 175 was 142.5, 538.5, and 1787.5 g·h/mL for the low-, mid-, and high-dose groups, respectively. The no-effect dose was 600 mg/kg. Females had statistically significantly lower (p 0.05) plasma AUC values than did males at all dose levels. At the NOEL (600 mg/kg/day), AUC0 24h values were 2369 M·h (509 g·h/mL) for males and 1985 M·h (427 g·h/mL) for females.

2.6.6.3.1.5. Rilpivirine: Non-Pivotal Oral Pilot Carcinogenicity Dose Range-Finding Studies in Mice

Oral studies in mice with TMC278 were conducted in preparation of the carcinogenicity study in that species. In a 2-week study with CD-1 mice, toxicity and exposure was determined, following administration by gavage (in aqueous HPMC) and via diet. In a 1-month study, the feasibility of the transgenic CB6F1-non TgrasH2 strain was tested. The final carcinogenicity dose range finding study was a 3-month study in CD-1 mice with administration by gavage.

2-Week Study with Swiss Mice

In a 2-week study with CD-1 mice (Tabulated Summary 2.6.7.6.A, TMC278-NC118), TMC278 was administered by gavage or via diet in doses up to 2000 and 5000 mg/kg/day, respectively.

No compound-related mortality was noted up to 400 mg/kg/day by either route. The high doses, 2000 (gavage) and 5000 mg/kg/day (diet), were not tolerated.

Almost exclusively at the highest tolerated dose of 400 mg/kg/day, either by gavage or diet, body weight gain was decreased up to 29%, whereas food consumption (diet administration only) was increased maximally 49%.

Hematology showed RBC, Hb, and HCT (all 8%) and increased reticulocyte count (23%) and reduced eosinophil counts in females when administered by diet (also at 40 mg/kg/day).



Increased serum concentrations were measured for total bilirubin (up to 3-fold at 400 mg/kg/day, 77% at 40 mg/kg/day), ALP (up to 3-fold), ALT (up to 3-fold), AST (72%), albumin (up to 22 %), total protein (13%), cholesterol (up to 48%), calcium (up to 8%), and urea (up to 40%) and decreased concentration of triglycerides (up to 73%).

At necropsy, increased liver weight was noted accompanied by pale, dark, and/or swollen aspect and associated with hepatocellular hypertrophy, marginal to slight focal necrosis, and marginal single cell necrosis, marginally increased mononuclear phagocytic system (MPS)-aggregates with necrotic hepatocytes, and prominent mitoses (females on diet, only).

Increased kidney weight was accompanied by pale and swollen aspect (females by gavage, only). Histopathology showed tubular dilatation and subchronic nephropathy and exudative nephritis.

Systemic exposure expressed as Cmax and AUC values determined at the end of the dosing period are given in Table 3 below (Tabulated Summary 2.6.7.6.A, TMC278-NC118 [TOX6479]).

Table 3. Exposure to TMC278 (Cmax and AUC) in a 2-Week Oral Study with TMC278 in Mice

Cmax (μg/mL) AUC0 24h (μg.h/mL) Dose(mg/kg/day) Route M (n = 3-9) F (n = 3-9) M (n = 3-9) F (n = 3-9)

40 diet 8.9 8.3 150 148

400 diet 57 63 1066 1320

5000 diet

400 gavage 63 94 687 1233

2000 gavage

M = males, F = females, Shaded = not sampled; dose not tolerated

1-Month Study with Transgenic Mice

In a 1-month study, TMC278 was administered orally by gavage to transgenic CB6F1-nonTgrasH2 mice at 0 (vehicle), 20, 80, or 320 mg/kg/day (Tabulated Summary 2.6.7.6.A, TMC278-NC121).

In the group dosed with 320 mg/kg/day, 2 mortalities occurred and signs of deteriorating clinical condition were noted. Almost exclusively at 320 mg/kg/day, body weight was decreased up to 13% and food consumption increased up to 22%.

Hematology showed reduced RBC, Hb, and HCT (all up to 6%); reduced reticulocyte count (56%, females only); and increased number of platelets (up to 24%).



Increased serum concentrations were determined for total bilirubin (3-fold, females), ALP (up to 3-fold), albumin (17%, males), total protein (up to 18%), cholesterol (up to 100%), calcium (27%, females), inorganic phosphate (42%, females), and urea (up to 72%).

At necropsy, increased weight of liver (up to 100% at 320 mg/kg/day, 29% at 80 mg/kg/day), spleen (up to 40%), and kidneys (14%, females) and decreased weight of thymus (73%, females) and ovaries (40%) was noted. Moreover, liver and spleen were enlarged and showed discoloration.

Upon histopathology, hepatocellular hypertrophy in all TMC278-treated groups was noted and at 320 mg/kg/day; this was associated with coagulative hepatocellular necrosis. Moreover, degenerative/necrotic nephropathy (also at 80 mg/kg/day); lymphoid depletion in thymus; myeloid cell hyperplasia in bone marrow; erythro/myelopoiesis in spleen; almost complete absence of ovulation; and uterus atrophy occurred.

Systemic exposure expressed as Cmax and AUC values are given in Table 4 below.

Table 4. Exposure to TMC278 (Cmax and AUC) in a 1-Month Oral Study with TMC278 in Mice

Cmax ( g/mL) AUC (μg.h/mL)aDose

(mg/kg/day) Day of Sampling M (n = 3) F (n = 3) M (n = 3) F (n = 3)

1 15 14 52 45 20

29 13 15 63 54

1 30 28 199 192 80

29 30 37 250 240

1 45 42 643 667 320

29 66 69 1090 942

a AUCinf: on Day 1 otherwise AUC0 24h, M: males, F: females



2.6.6.3.1.6. Rilpivirine: 3-Month Oral Toxicity and Carcinogenicity Dose Range Finding Study with TMC278.HCl in Mice

TMC278 was administered once daily by oral gavage for 3 months to CD-1 mice. Groups of 25 animals per sex (10 for the main study and 15 satellite animals for toxicokinetics) were given 0 (vehicle; 12 satellite animals per sex), 20, 80, or 320 mg/kg/day in 10 mL/kg (Tabulated Summary 2.6.7.7.E, TMC278-NC119 [TOX6739]). All animals were observed regularly for clinical signs, morbidity, and mortality. Body weight of all animals was recorded, but assessed for main animals only. For main animals, food consumption and body weight gain were determined. Main animals were sampled for hematology and serum chemistry, and necropsied and inspected for gross lesions at the end of the study. A range of organs was weighed and a complete set of organs and tissues was preserved for microscopic examination. For possible electron microscopic evaluation, liver samples from 5 male and 5 female mice of the control group and the group treated with 320 mg/kg/day were fixed in phosphate buffer containing 3% glutaraldehyde. Blood samples for toxicokinetic analysis were drawn from 3 satellite animals per gender per time point at 5 time points within 24 hours on Days 1, 31, and 87. After the last sampling these animals were killed.

There were no mortalities. No clinical signs were noted, except abdominal distention in 5/10 males and 7/10 females dosed with 320 mg/kg/day from Week 6 of treatment onwards.

Body weight at the end of the dosing period was increased up to 11% in the males and females dosed with 320 mg/kg/day. This increase was associated with a body weight gain over the whole dosing period of slightly more than 150%. The effects on body weight and body weight gain were in line with a 20% higher food consumption essentially throughout the dosing period in males and females dosed with 320 mg/kg/day.

Red blood cell count, Hb, and HCT were 10% lower in males and females dosed with 320 mg/kg/day. In females, this reduction in RBC parameters was associated with a 37% increase in reticulocyte count. In males treated with 320 mg/kg/day, leukocyte count was only 53% of control value. Lymphocyte, eosinophil, and to a lesser extent neutrophil counts contributed to this lower value.

Alkaline phosphatase and ALT activities in serum were more than 2-fold higher in males and females dosed with 320 mg/kg/day. Also in this group, females showed a 25% increase in urea. In females dosed with 80 and 320 mg/kg/day, cholesterol had increased 29% and 42%, respectively, whereas triglycerides had decreased almost 40% and more than 60%, respectively. Total protein and albumin had increased by 6% to 14% predominantly in females given 80 or 320 mg/kg/day. Calcium and inorganic phosphate had increased 7% to 18% in animals dosed with 320 mg/kg/day.



Liver weight was increased in a dose-related fashion in animals treated with 80 (20% and 37% in males and females, respectively) and 320 mg/kg/day (2.1- and 2.7-fold higher in males and females, respectively). Mean spleen weight in females treated with 320 mg/kg/day was 167% of control value. This increase was not statistically significant, but correlated to the swollen aspect noted in 3 out of 10 females in this group and the extramedullar hematopoiesis upon microscopic evaluations.

Histopathology confirmed that the liver was the major target for TMC278. The most prominent effect was hepatocellular hypertrophy with a dose-related increase in incidence and severity in the animals dosed with 80 and 320 mg/kg/day. In animals given the higher dose, this effect was associated with hepatocellular vacuolization, single cell necrosis, and pigmentation and proliferation of Kupffer cells; all to a slight to moderate degree. Electron microscopy revealed peroxisome proliferation in the animals dosed with 320 mg/kg/day.

In half the number of the female mice treated with 320 mg/kg/day, minimal to moderate nephropathy was noted. The nephropathy was characterized by slight to marked multifocal tubular basophilia; minimal to slight glomerulopathy (atrophic glomeruli with thickened Bowman’s capsule amidst basophilic tubules); minimal to moderate mononuclear cell infiltration; minimal to slight interstitial fibrosis; minimal tubular dilatation; and slight cortical mineralization.

At 320 mg/kg/day, a marginally increased incidence was noted of swollen cells and/or cells with dense cytoplasm in the adrenal zona fasciculata in males, whereas females showed a marginal decrease of a clear X-zone with increased brown degeneration in that zone.

In females treated with the high dose, a marginal decrease of the number of corpora lutea and generations of corpora lutea was noted in ovaries. Also, granulocyte infiltration in the endometrium was marginally decreased in this group. It cannot be excluded that these gonadal effects indicate a reduced cyclic activity.

Moreover, animals dosed with 320 mg/kg/day showed extramedullar hematopoiesis in liver (marginal) and spleen (slight to moderate), and slight to moderate increase of the myeloid/erythroid ratio in bone marrow. These effects are likely associated with the effects on RBC parameters.

Based on the findings in animals with 80 mg/kg/day in liver, the NOAEL was considered to be 20 mg/kg/day. Systemic exposure expressed as Cmax and AUC values are given in Table 5 below.



Table 5. Exposure to TMC278 (Cmax and AUC) in a 3-Month Oral Study with TMC278 in Mice


(mg/kg/day) Day of

Sampling M (n = 15) F (n = 15) M (n = 15) F (n = 15)

1 14 13 71 59

31 14 18 61b 74 20

87 18 19 80 61

1 28 32 236 250

31 38 37 263 313 80

87 34 42 210 313

1 63 55 1010 707

31 63 84 860 1170 320

87 61 90 665 1360

a AUCinf on Day 1 and AUC0 24h on Days 31 and 87, M: males, F: females, b: AUC0 8h.

2.6.6.3.1.7. Tenofovir DF: A 14-Day Repeat Dose Oral Toxicity Study of Tenofovir DF in ICR CD-1 Mice (M990191)

The purpose of this study was to investigate the potential toxicity of TDF during daily oral (gavage) administration to the mouse for 13 consecutive days (Tabulated Summary 2.6.7.6.B,M990191). Male and female mice (ICR CD-1), 5/sex/group, were dosed with 100, 300, or 1000 mg/kg of TDF by oral gavage for 13 days. Animals were generally monitored BID for adverse clinical signs. Live-phase assessment included assessment of activity, posture, respiration, hydration status, and overall body condition. Body weights were taken daily through Day 4 of the study and then every other day. Hematology and clinical chemistry analytes were evaluated on blood samples and histologic evaluation was conducted on selected tissues collected at necropsy on the fourteenth day of the study.

There was 1 mortality in the 1000 mg/kg/day group; a test article-related effect could not be excluded. There was a mild, non dose-dependent reduction of the absolute neutrophil count (25% to 33%) in all TDF-treated groups. Two of 8 mice in the 1000 mg/kg/day group, both females, had a marked reduction of their platelet counts. There was a dose-related rise of the mean serum ALT activity (1.3- to 2-fold) in all TDF-treated groups. In the 300 and 1000 mg/kg/day groups, the mean ALP activity was raised 1.2- and 1.6-fold, respectively, and the mean phosphorus concentration was raised approximately 7% and 15%, respectively. Microscopic alterations included a minimal increase in the incidence of single-cell necrosis and centrilobular hypertrophy in the liver and minimal renal proximal tubular epithelial karyomegaly in the 1000 mg/kg/day group. Based on the findings of this study, the NOEL was considered to be 100 mg/kg/day.



2.6.6.3.1.8. Tenofovir DF: A 13-Week Oral Gavage Toxicity Study of Tenofovir DF in the Albino Mouse (M990203)

The purpose of this study was to investigate the potential toxicity of TDF at doses of 0 (vehicle), 100, 300, or 1000 mg/kg/day (reduced to 600 mg/kg/day on Day 9) during daily oral gavage administration to mice (15/sex/group) for 13 consecutive weeks (Tabulated Summary 2.6.7.7.F, M990203). Toxicokinetics were assessed in the 100 and 300 mg/kg/day (32M/group) and 1000/600 mg/kg/day group (32/sex/group plus 16/sex/group for the Day 1 600 mg/kg/day dosage). Clinical observations (BID), physical examination (once weekly), body weight (weekly), food consumption (weekly), ophthalmology (pretreatment and Week 13), hematology, clinical biochemical analysis (Week 13), and gross pathology and organ weights (termination only) were determined for all animals scheduled for necropsy. Histopathologic examination was conducted on tissues from Groups 1 and 4, and all gross lesions and target organs from Groups 2 and 3.

There were no test article-related deaths recorded during the 13-week treatment period. During the first few days of treatment, there were 13 unscheduled deaths recorded for animals receiving 1000 mg/kg/day. Prior to death, the animals had decreased activity and/or a swollen abdomen. Following consultation with the sponsor, the dose level for Group 4 was reduced on Day 9 from 1000 to 600 mg/kg/day. The deaths seen at 1000 mg/kg/day were considered to be due to problems administrating the test suspension and not test article-related. During the process of administering the test suspension, the gavage needle became blocked on a number of occasions, resulting in additional stress for the animal. It is for this reason that 600 mg/kg/day (60 mg/mL) is considered to be the highest dose level of TDF that may be practically administered to albino mice. There were no clinical signs recorded during the 13-week treatment period that were considered to be treatment-related. There were no effects on body weight, food consumption, ophthalmology, hematology, or biochemistry parameters. There were no treatment-related effects on organ weight or macroscopic findings. Microscopic findings associated with treatment were seen in the kidneys and duodenum. Minimal tubular karyomegaly was seen in males receiving 300 and 600 mg/kg/day and in females receiving 100, 300, and 600 mg/kg/day. Epithelial hypertrophy was also recorded for animals receiving 300 and 600 mg/kg/day.

In conclusion, the oral gavage administration of TDF at dosages of 0, 100, 300, or 600 mg/kg/day to mice for 13 weeks was well-tolerated, and 600 mg/kg/day was considered to be the highest practical dose for administration by oral gavage to mice for studies of a longer duration. However, a NOEL was not determined due to the finding of renal tubular karyomegaly at the lowest dose of 100 mg/kg/day in this study.



Pharmacokinetic Evaluation, M990203-PK

Concentrations of TFV were determined in plasma samples obtained during a 13 week repeated dose toxicity study after oral gavage of TDF (GS-4331-05) in albino mice (Tabulated Summary 2.6.5.4.Z, M990203-PK). Three groups of mice designated for toxicokinetic analysis received daily oral doses of TDF at 100, 300, and 1000 mg/kg/day for 13 weeks (14/sex/group). The dose level of the 1000 mg/kg/day dose was reduced to 600 mg/kg/day on Day 9 and for the remainder of the study due to problems attributed to the administration of the test suspension. Plasma was drawn from male mice at all dose levels, and from male and female animals in the 600- and 1000-mg/kg/day dose cohorts.

Blood samples were obtained for pharmacokinetic analyses on Days 1 and 91. Plasma samples were assayed for TFV using a validated reversed-phase ion-pair HPLC method with fluorescence derivatization. In general, mean TFV plasma concentration values from 2 animals sampled at each time point were analyzed; however, single values were used when 2 values were not available.

No statistical analysis could be performed on the limited data, therefore differences could not be discerned between days and gender. Overall, TFV plasma Cmax and AUC appeared to increase in a reasonably dose proportional manner. Results on Day 1 were as follows: Cmax = 3.38, 5.89, 22.7, and 35.1 g/mL and AUCinf = 51.3, 46.3, 25.5, and 64.5 g·h/mL for the 100, 300, 600, and 1000 mg/kg/day dose groups, respectively. The AUC values from Day 1 were all associated with an unacceptably high percent of the values extrapolated (19% to 84%). Following the Day 91 dose, Cmax values were 8.82, 17.9, and 33.8 g/mL and AUCtau values were 14.6, 35.8, and 61.7 g·h/mL for the 100-, 300-, and 600-mg/kg/day dose groups, respectively.

2.6.6.3.2. Rat

2.6.6.3.2.1. Emtricitabine: TP-0006: A 3-Month Oral Gavage Study for Bioassay Dose Selection in CD Rats (TOX097)

To evaluate the subchronic toxicity in rats of FTC and help choose doses for a 2-year carcinogenicity bioassay, Charles River CD rats (10/sex/dose) were given 0, 120, 600, or 3000 mg/kg/day for 3 months, euthanized, and necropsied (Tabulated Summary 2.6.7.7.G,TOX097). An additional 18 rats/sex/dose were given the same doses to evaluate systemic exposure after the second and ninetieth doses. During the dosing period, rats were evaluated for clinical signs of toxicity, and body weight and food consumption were measured. Urine samples were collected for urinalysis at the end of the dosing period. At necropsy, blood was collected for clinical pathology studies; organs were weighed; rats were examined for gross pathologic lesions; and tissues were collected for light microscopic examination.



Rats tolerated daily FTC doses up to 3000 mg/kg/day for 3 months without clinical signs of toxicity or effects on body weight gain, food consumption, or clinical chemistry parameters. Mild anemia was noted at 3000 mg/kg/day. No gross pathologic or histopathologic lesions occurred. Pituitary and thyroid glands weighed less at 3000 mg/kg/day, but there were no histopathologic changes to correlate with the reduced weights.

Emtricitabine was rapidly absorbed, with females absorbing the compound more rapidly than males (Tmax = 1–2 hours). Day 90 Cmax was 20% to 50% greater in females than males. Day 90 mean Cmax was greater than Day 2 Cmax for both sexes (5% to 30% in males, 20% to 60% in females). Day 90 mean AUC0 24h was 20% to 40% greater than Day 2 AUC0 24h in females, but about the same as Day 2 AUC0 24h in males. Mean t1/2 was 2.4 to 3.8 hours on Day 2 and 3.6 to 6.5 hours on Day 90. Dose-normalized Cmax tended to decrease with increasing dose, but dose-normalized AUC0 24h remained relatively constant, suggesting a linear relationship between systemic exposure and daily dose of FTC in rats from 120 to 3000 mg/kg/day.

2.6.6.3.2.2. Rilpivirine: Non-Pivotal Pilot Oral Toxicity and Dose Range Finding Carcinogenicity, and Free Base – Salt Bridging Studies

For RPV, the pivotal studies with administration durations ranging from 1 to 6 months were preceded by pilot studies. Following the selection of the HCl-salt for further final development, a 1-month bridging study with TMC278 base and TMC278 was done. Two 2-week studies with TMC278 base and TMC278 administered by gavage or via diet were done for the selection of the test article and the route of administration in the oral carcinogenicity study.

5-Day Oral Tolerance Study with TMC278

TMC278 base dissolved in PEG400 was administered once daily by oral gavage for 5 days at 0 (vehicle), 40, or 400 mg/kg/day in a dose volume of 8 mL/kg to 5 male Sprague Dawley rats/group (Tabulated Summary 2.6.7.6.A, TMC278-Exp5463; Tabulated Summary 2.6.7.2.B, FK4103 [Exp5463]).

There was no mortality and treatment was well tolerated. There were no relevant clinical signs or effects on body weight or on hematology. There were no relevant histopathological changes in the examined tissues. Systemic exposure after repeated oral dosing of 400 mg/kg/day was shown by a Cmax of 2 μg/mL and an AUCinf of 28 g.h/mL.

2-Week Oral Toxicity Study with TMC278 Base

TMC278 base dissolved in PEG400 + CA was administered once daily, by oral gavage, for 2 weeks to Sprague Dawley rats at 0 (water), 0 (vehicle), 40, 120, or 400 mg/kg/day in 10 mL/kg (Tabulated Summary 2.6.7.6.A, TMC278-Exp5535, Tabulated Summary 2.6.7.2.B, FK4243 (Exp5535). All groups comprised 5 male and 5 female rats for the toxicological evaluations and the groups treated with TMC278 base had an additional 4 males and 4 females for toxicokinetic analysis.



There were no mortalities in this study.

Increased serum concentrations of total protein (3%, males) and albumin (7%, males) were noted in animals dosed with 400 mg/kg/day. Moreover, decreased concentrations of thyroxine (T4; maximally 66%) were determined at all doses in males and/or females associated with increased thyroid stimulating hormone (TSH; up to 100%) in animals dosed with 120 and 400 mg/kg/day.

At necropsy, increased thyroid gland weight (19%) in females given 400 mg/kg/day and hypertrophy of the thyroid follicular epithelium in male and/or female rats at all doses were noted.


Table 6. Exposure to TMC278 (Cmax and AUC) in a 2-Week Oral Study with TMC278 Base in Rats


(mg/kg/day) Day of

Sampling M (n = 4) F (n = 4) M (n = 4) F (n = 4)

1 1.6 3.2 13 29 40

14 2.2 5.7 16 42

1 2.8 4.7 30 55 120

14 3.6 7.8 35 88

1 10 14 86 128 400

14 8.4 15 84 152


2-Week Oral Study with TMC278 and TMC278.HCl by Gavage and via Diet

TMC278 was administered once daily to Sprague Dawley rats by oral gavage suspended in 0.5% (m/v) aqueous HPMC at a dose of 400 mg/kg/day or by dietary administration at 400 or 1200 mg/kg/day (Tabulated Summary 2.6.7.6.A, TMC278-NC136). TMC278 base was administered by diet at 400 mg/kg/day. The control group received unmedicated diet. All groups comprised 5 male and 5 female rats for the toxicological evaluations and the groups treated with TMC278 base had additionally 6 males and 6 females for toxicokinetic analysis.

No mortality was noted during this study. There were no toxicologically relevant differences between groups dosed with TMC278 base or TMC278 at 400 mg/kg/day by gavage or through the diet.

With both forms, decreased WBC count (maximally 23%), and decreased Hb and HCT (maximally 6%) occurred.



Increased ALT activity in serum (up to 44%) in males treated with 400 mg/kg/day and decreased concentration of cholesterol (29%) in males given TMC278 by gavage were determined.

Microscopy showed increased diffuse thyroid follicular hypertrophy; an increase in the number of swollen/vacuolated cells in the pars distalis of the pituitary gland in male rats; and hepatocellular hypertrophy in male and female rats at 1200 mg/kg/day.


Table 7. Exposure to TMC278 (Cmax and AUC) in a 2-Week Oral Study in Rats with TMC278 Base and TMC278

Cmax ( g/mL) AUC0 24h ( g.h/mL) Dose(mg/kg/day) Form Route

Days of Sampling M (n = 6) F (n = 6) M (n = 6) F (n = 6)

400 Base Diet 12 3.7 4.3 57 86

400 Salt Diet 12 5.2 7.2 82 137

400 Salt Gavage 12 7.3 12 51 103

1200 Salt Diet 12 11 13 180 266

M: males, F: females

2-Week Oral Pilot Carcinogenicity Dose Range Finding Study with TMC278.HCl in Rats

TMC278 suspended in 0.5% (m/v) aqueous HPMC was administered once daily, by oral gavage, for 2 weeks to Sprague Dawley rats at 0 (vehicle), 400, 1500, or 2000 mg/kg/day in 10 mL/kg (Tabulated Summary 2.6.7.6.A, TMC278-NC177). All groups comprised 5 male and 5 female rats for the toxicological evaluations and the groups treated with TMC278 had additionally 3 males and 3 females for toxicokinetic analysis.

There were no mortalities in this study.

Increased activated partial thromboplastin time (APPT) and prothrombin time (PT, both up to 20%) were determined in males from 1500 mg/kg/day and above.

Serum chemistry showed decreased serum concentrations of chloride (3%), glucose (up to 18%), urea nitrogen (33%), and creatinine (maximally 23%) in females given 1500 or 2000 mg/kg/day. In the latter group, males had decreased concentrations of cholesterol (30%).

Increased urinary volume (up to 100%) was recorded in animals given 1500 and 2000 mg/kg/day.



At necropsy, increased weight of the thyroid gland in females of all groups treated with TMC278 was noted in association with hypertrophy of follicular cells. In males given 2000 mg/kg/day, hypertrophy of follicular cells was associated with multifocal vacuolated cells in the pars distalis of the pituitary gland.


Table 8. Exposure to TMC278 (Cmax and AUC) in a 2-Week Oral Study in Rats with TMC278

Cmax (μg/mL) AUC (μg.h/mL)aDose(mg/kg/day) Day of Sampling M (n = 3) F (n = 3) M (n = 3) F (n = 3)

1 5.4 12 50 78 400

14 5.2 12 42 96

1 18 12 153 152 1500

14 9.5 12 86 115

1 10 17 103 206b 2000

14 7.7 14 77 147

a AUCinf: on Day 1 otherwise AUC0 24h, b: n=2, M: males, F: females.

1-Month Bridging Study with TMC278 Base and TMC278 in Rats

This study served to bridge the toxicity and toxicokinetics after administration of TMC278 base with those after TMC278 (Tabulated Summary 2.6.7.6.A, TMC278-NC117). Both compounds were administered once daily, by oral gavage, to Sprague Dawley rats for 4 weeks at 0 (vehicle), 10, or 400 mg/kg/day in 10 mL/kg. TMC278 base was dissolved in PEG400 + CA and TMC278 was suspended in 0.5% (m/v) aqueous HPMC. All groups comprised 10 male and 10 female rats for the toxicological evaluations and the groups treated with TMC278 base and TMC278 had additionally 6 males and 6 females for toxicokinetic analysis.

No compound related mortality was noted. No effects were noted in either group dosed with 10 mg/kg/day. There were no significant differences in the toxicity parameters of both forms dosed at 400 mg/kg/day.

Hematology showed decreased HCT (3%) and increased RBC count (4%).

Increased serum concentrations of albumin (up to 7%, males) and total protein (5%, males), and decreased levels of cholesterol (up to 22%, females), triglycerides (35%, females), urea (up to 18%), potassium (13%, females), and chloride (2%) were determined. Moreover, serum concentration of T4 was decreased up to 61%, whereas those of triiodothyronine (T3) and TSH were increased up to 57% (males only) and up to 51%, respectively.

Urinary volume was increased up to 98%.



At necropsy, increased weight of thyroid gland (up to 60%, females) and liver (up to 24%) were recorded. Hepatocellular hypertrophy in liver, diffuse follicular hypertrophy in thyroid gland, and an increase in swollen/vacuolated cells in the pars distalis of the pituitary gland was noted.


Table 9. Exposure to TMC278 (Cmax and AUC) in a 1-Month Oral Bridging Study with TMC278 Base and TMC278 in Rats

Cmax (μg/mL) AUC (μg.h/mL)aTMC278 Base (mg/kg/day) Sampling Day M (n = 6) F (n = 6) M (n = 6) F (n = 6)

1 0.87 1.5 6.3 10 10

24 0.74 1.4 5.4 8.6

1 8.3 12 101 123 400

24 7.7 13 90 149

TMC278 (mg/kg/day)

1 0.90 1.6 5.5 8.6 10

24 0.76 1.7 4.5 7.8

1 7.0 9.5 52 100 400

24 4.8 9.0 33 86

a AUCinf: on Day1 otherwise AUC0 24h, M: males, F: Females

2.6.6.3.2.3. Rilpivirine: 1-Month Oral Toxicity Study Including Immunotoxicity Evaluation in Rats (Pivotal Study)

TMC278 base formulated in PEG400 + CA was administered once daily, by oral gavage, for 4 weeks to groups of 18 male and 18 female Sprague Dawley rats at 0 (water, negative control), 0 (vehicle), 10, 40, or 160 mg/kg/day in 10 mL/kg (Tabulated Summary 2.6.7.7.H, TMC278-Exp5692); 8 male and 8 female satellite animals per group were used for the plaque forming cells (PFC) assay. TMC278-treated groups had 4 more males and females for toxicokinetic analysis. Regular observations were made for clinical signs, morbidity, and mortality. Body weight, food consumption, and body weight gain were determined throughout the dosing period. Ophthalmic examinations were performed prior to dosing and at the end of the dosing period (negative control, vehicle, and 160 mg/kg/day animals only). All main animals were sampled for hematological, serum chemistry, and urinalysis parameters at the end of the dosing period. The PFC satellites received on 4 consecutive days an intravenous injection with sheep RBCs. Thereafter, they were killed and a suspension of spleen cells was prepared and incubated with sheep RBCs and guinea pig complement. The resulting plaques are considered to be antibody-producing cells. At necropsy, gross lesions and organ weight were recorded, and tissues and organs were sampled for histopathology. Blood samples were drawn for toxicokinetics on Days 1 and 29.



There were no mortalities associated with TMC278, relevant clinical signs, or effects on body weight or food consumption; no treatment-related ophthalmic effects; and no effects on the direct PFC assay. A decrease in glucose (10%) was seen in females given 160 mg/kg/day. Higher thyroid gland weights (maximally 25%) and liver weights (maximally 23%) compared to the vehicle group were recorded in the groups dosed with 40 and 160 mg/kg/day. The weight of the pituitary gland was slightly increased in males (16%) and females (14%) dosed with 160 mg/kg/day. The increase in thyroid gland weight was associated with minimal follicular hypertrophy in the majority of the males and 4 out of 10 females given 160 mg/kg/day and in 2 out of 10 males given 40 mg/kg/day.

In view of the absence of findings of toxicological relevance in the group treated with 10 mg/kg/day, this dose was the NOAEL. Moreover, the absence of effects in the PFC assay indicated that TMC278 has no immunotoxic potential. Systemic exposure expressed as Cmax and AUC values is given in Table 10 below.

Table 10. Exposure to TMC278 (Cmax and AUC) in a 1-Month Toxicity Study of TMC278 Base in Rats

Cmax (μg/Ml) AUC (μg.h/Ml)aDose

(mg/kg/day) Sampling

Day M (n = 4) F (n = 4) M (n = 4) F (n = 4)

1 0.94 2.0 7.5 14 10

29 0.88 1.6 7.2 14

1 2.0 3.2 23 30 40

29 2.6 5.8 27 42

1 6.5 9.6 52 77 160

29 6.7 8.8 51 89




2.6.6.3.2.4. Rilpivirine: 6-Month Oral Toxicity Study Including 1-Month Recovery in Rats

TMC278 base formulated in PEG400 + CA was administered once daily, by oral gavage, for 6 months to groups of 20 male and 20 female Sprague Dawley rats (Tabulated Summary 2.6.7.7.I, TMC278-NC101) at 0 (vehicle), 40, 120, or 400 mg/kg/day in 10 mL/kg. There were 6 satellite rats of each sex per TMC278-treated group for toxicokinetic analysis, and 10 rats of each sex in the control and high dose group for assessment of reversibility of the findings at 1 month postdose. Regular observations were made for clinical signs, morbidity, and mortality. Body weight, food consumption, and body weight gain were determined throughout the dosing and recovery periods. Ophthalmic examinations were performed prior to dosing and at the end of the dosing and recovery. Samples for hematological, serum chemistry, and urinalysis parameters were taken after 3 months of treatment and at the end of dosing and recovery periods. In addition, serum concentrations of TSH, T3, T4, adrenocorticotropic hormone (ACTH), corticosterone, and progesterone were determined in samples taken prior to the autopsies. At necropsy, gross lesions and organ weight were recorded and tissues and organs were sampled for histopathology. Blood samples were drawn for toxicokinetics on Days 1, 31, and 87. At necropsy, blood and liver samples were taken for determination of TMC278.

Increasing difficulties with the daily administration by gavage, morbidity, and deaths started to occur after the first 2 months of dosing with comparable incidences in all groups including controls. For this reason, the dosing regimen was changed from once daily to BID dosing (5 mL/kg with an interval of 1.5 hours) from Day 84 onwards.

The cause of death (reaching 50% in some of the groups) was concluded to be associated with the relatively high volume and viscosity of the formulation, necessitating a long gavage time during which the animals physically opposed the manual restraint. This led to gavage errors, regurgitation, and restrain trauma. In the 1-month postdosing period, no further mortalities occurred.

Compound-related clinical signs included salivation in females at all dose levels and in males at 400 mg/kg/day, and wet urogenital region in both genders at 400 mg/kg/day. There were no relevant effects on ophthalmic examinations, body weight, or food consumption.

In hematology, increases in APTT (up to 23%) and PT (up to 27%) were noted in TMC278-treated males of all groups, without a dose-related trend, at all sampling times, including the end of the recovery period. At the end of the dosing period, RBC count, Hb, and HCT of males dosed with 400 mg/kg/day were reduced by 5%. The eosinophil count in TMC278-treated females were 25% to 33% lower than control values without a clear dose-related trend. Red blood cell parameters and eosinophil counts showed complete recovery after termination of dosing.



Serum chemistry after 3 months of treatment and at the end of the dosing period showed increases in total protein (4%, in females at 400 mg/kg/day), and albumin (up to 10%, at 120 and 400 mg/kg/day). Decreases up to approximately 40% were recorded in triglycerides and total bilirubin levels in all TMC278-treated groups, without a clear relationship to dose. Alkaline phosphatase displayed a dose and treatment-duration related increase in males at 120 and 400 mg/kg/day. The increase was 30% and 53%, respectively, after 3 months of treatment and 32% and 70%, respectively, at the end of the dosing period. Inorganic phosphate was slightly higher (maximally 11%) after 3 months of dosing in females at all dose levels (dose-related) and in males at 120 and 400 mg/kg/day. Increases in urea (10%, in males at 120 and 400 mg/kg/day) and creatinine (12%, males of all TMC278-treated groups without a dose relationship) were noted at the end of treatment. All serum chemistry changes showed complete reversibility at the end of the recovery period.

Urinalysis showed no differences between the treated groups and the control group. The 2.5-fold higher concentration in urinary ketones at the end of the 1-month recovery period in males treated with 400 mg/kg/day is not likely to be treatment-related.

Thyroid stimulating hormone levels in serum showed a more or less dose-related increase from 45% and 77% in males and females, respectively, in the group dosed with 40 mg/kg/day, and up to 23% and 2.5-fold, respectively, in the group treated with 400 mg/kg/day. This effect was associated with a similarly statistically significant and dose-related decrease of serum T4 ranging from 36% and 25%, respectively, in males and females dosed with 40 mg/kg/day, and up to 45% and 46% at the 400 mg/kg/day dose. In contrast, T3 showed lesser and equivocal effects: 30% decrease in males dosed with 40 mg/kg/day and 34% increase in males dosed with 400 mg/kg/day. At the end of the postdosing period, no parameters, except T4, showed differences with control values. Thyroxine levels in females treated with 400 mg/kg/day were 138% of control value, indicating that thyroid homeostasis had not yet completely recovered.

Corticosterone, progesterone, and ACTH levels were highly variable. The overall trend indicated a decrease of corticosterone levels and an increase in ACTH and progesterone concentrations in the groups dosed with 120 and 400 mg/kg/day. No trend of any effect was observed at the end of the postdosing period.

At necropsy, liver weight was increased without a clear dose relationship in animals treated with 120 and 400 mg/kg/day. Thyroid gland weight of animals in all groups was increased. The increase was dose-dependent ranging from 20% to 50% and was statistically significant in all groups. Reversibility of the effect in females was not complete at 1 month after dosing as the weight of the organ was still 40% higher than controls.



Livers of the animals treated with 400 mg/kg/day showed pronounced lobulation. Upon microscopy, a dose-related increase of hepatocellular hypertrophy was noted in the animals of the groups treated with 120 and 400 mg/kg/day. This hypertrophy was associated with a decrease in hepatocellular necrosis and hemorrhages, and in females with a decrease of vacuoles physiologically associated with fatty storage. The hepatocellular hypertrophy was considered recovered at the end of the 1-month postdosing period. The histopathological findings and recovery are in line with the serum chemistry effects and the effects on liver weight.

In the thyroid gland, a dose-related increase of diffuse follicular hypertrophy was observed in males and females of all groups. This hypertrophy was associated with an increase in the number of small follicles. In addition, unilateral focal follicular hyperplasia was noted in 1 animal dosed with 40 mg/kg/day and 1 animal dosed with 120 mg/kg/day, and in 3 animals dosed with 400 mg/kg/day. The follicular hypertrophy and small follicles were still noted in the animals allowed to recover from treatment with 400 mg/kg/day for 1 month, although to a lesser extent and incidence than in the animals killed at the end of the dosing period. The histopathological findings and incomplete recovery are in line with the thyroid-associated endocrinology data and the effects on the weight of the organ. In the pituitary gland of males from all groups, the number of swollen/vacuolated cells in the pars distalis was increased. These cells are known to produce TSH {15704}. This effect was not noted in animals killed at the end of the postdosing period, indicating complete recovery. This finding and its complete recovery are in line with the effects on TSH levels and the effects at the thyroid gland level.

In 20% to 25% of the males and females treated with 400 mg/kg/day, the macrophages that spontaneously form aggregates in the mesenteric lymph nodes had a swollen-vacuolated appearance. This effect was also noted in 30% to 33% of the animals at the end of the postdosing period.

In view of the effects on coagulation parameters and the thyroid and pituitary glands observed in the low dose group treated with 40 mg/kg/day, a NOAEL could not be established in this study. Systemic exposure expressed as Cmax and AUC values are given in Table 11 below.



Table 11. Exposure to TMC278 (Cmax and AUC) in a 6-Month Oral Toxicity Study with TMC278 Base in Rats

Cmax (μg/Ml) AUC (μg.h/Ml)aDose(mg/kg/day) Sampling days M (n = 3) F (n = 3) M (n = 3) F (n = 3)

1 2.9 6.5 19 32

84 3.4 8.2 19 41 40

175 1.7 6.6 12 50

1 6.4 8.5 53 83

84 3.4 11 41 100 120

175 3.0 8.8 35 116

1 9.1 17 92 160

84 3.9 15 57 184 400

175 6.2 16 73 244

a AUCinf: on Day 1 otherwise AUC0 24h, M: male, F: female

2.6.6.3.2.5. Tenofovir DF: 5-Day Repeated Dose Oral Toxicity Study of GS-4331-02 in Male Sprague-Dawley Rats (non-GLP) (96-TOX-4331-002)

The toxicities of GS-4331-02, an oral CA prodrug salt of TFV were evaluated in male Sprague-Dawley rats (4/group) following once daily oral gavage dosing (20 mL/kg) for 5 consecutive days at doses of 0, 25, 100, or 400 mg/kg/day (Tabulated Summary 2.6.7.6.B,96-TOX-4331-002). The control group received sterile 50 mM CA (pH 2.2). Based on the reduced terminal body weights and body weight gains in the 400 mg/kg/day group, the NOEL for GS-4331-02 administered to rats via oral gavage once daily for 5 consecutive days is equal to or greater than 100 mg/kg/day and less than 400 mg/kg/day.

2.6.6.3.2.6. Tenofovir DF: A 14-Day Oral Gavage Toxicity Study of bis-POC PMPA Fumarate (GS-4331-05; PMPA Prodrug) in the Albino Rat (98-TOX-4331-004)

The purpose of this study was to investigate the potential toxicity of TDF (bis-POC PMPA Fumarate; GS-4331-05; PMPA Prodrug) in male rats following daily oral administration for 14 days (Tabulated Summary 2.6.7.7.J, 98-TOX-4331-004). Four groups, each of 5 males, received TDF at dose levels of 500, 750, 1000, or 1250 mg/kg/day. A fifth group of 5 males received the vehicle (suspension vehicle) and acted as the control. Eight animals were found dead or euthanized moribund prior to schedule termination: 1 rat dosed at 750 mg/kg/day (Day 11), 2 rats dosed at 1000 mg/kg/day (Day 8 or 11), and 5 rats dosed at 1250 mg/kg/day (Day 8 or 9). Lower body weight/body weight gains were recorded in animals receiving 500 mg/kg/day. The mean AST and ALT activities were increased 1.3-fold and 2.5-fold, respectively, at 750 and 1000 mg/kg/day (no clinical chemistry data for the 1250 mg/kg/day group). There were slight increases in adrenal weights and decreases in



thymus weights for the 750- or 1000-mg/kg/day groups. Gross necropsy findings considered possibly drug related included stomach dilatation; raised and/or depressed areas (1000 and 1250 mg/kg/day); stomach mucosal thickening (1000 mg/kg/day); areas of gastric discoloration (1250 mg/kg/day); duodenal thickening (750 and 1000 mg/kg/day); dilatation of the ileum and jejunum (1250 mg/kg/day); small spleens (750 and 1250 mg/kg/day); small thymuses ( 750 mg/kg/day); and emaciated carcass (1250 mg/kg/day). Treatment-related histopathologic lesions included inflammation of the stomach, duodenum, ileum, and/or jejunum ( 750 mg/kg/day); erosion and/or ulceration of the glandular and nonglandular stomach ( 1000 mg/kg/day); stomach hyperplasia ( 750 mg/kg/day); duodenum glandular dilatation (1250 mg/kg/day); and duodenum mucosal erosion/hyperplasia ( 750 mg/kg/day). Lymphoid atrophy of the spleen and thymus ( 750 mg/kg/day) were observed. The NOAEL for repeated doses of TDF in rats is considered to be 500 mg/kg/day following daily oral gavage treatment for 14 consecutive days.

Pharmacokinetic Evaluation (98-TOX-4331-004-PK)

The pharmacokinetic portion of study 98-TOX-4331-004 consisted of 5 groups with 5 rats per group divided into control, 500, 750, 1000, and 1250 mg/kg/day TDF. Plasma samples were collected from animals 1 hour following test article administration on days 1 and 14 (Tabulated Summary 2.6.5.4.GG). Plasma samples were analyzed for TFV concentration by a validated HPLC assay following fluorescence derivatization. All animals in dose groups that received TDF demonstrated exposure to TFV based on measurable levels of TFV in plasma on days 1 and 14. Plasma TFV concentrations on day 1 were not proportional with dose, suggesting saturation of drug absorption.

2.6.6.3.2.7. Tenofovir DF: A 28-Day Oral Gavage Toxicity Study GS-4331-05 in the Albino Rat (96-TOX-4331-003)

The objective of this study was to investigate the potential toxicity of TDF in Sprague--Dawley rats (l0/sex/group) dosed via oral gavage once daily for 28 consecutive days at 0 (vehicle), 20, 100, or 500 mg/kg/day (Groups 1 to 4, respectively) (Tabulated Summary 2.6.7.7.K, 96-TOX-4331-003). Toxicokinetic-only cohorts (8M/Groups 2, 3, 4; 8F Group 4) had blood samples taken on study Days 1 and 28 to assess systemic drug exposure. Toxicity associated with test article administration was evaluated by monitoring clinical signs of toxicity (BID); body weights and food consumption (weekly); clinical chemistry and hematology values (at study termination); and gross necropsy findings, organ weight changes, and histopathologic effects in protocol-selected tissues.

One animal was found dead on Day 12 of the treatment period. Gross pathological findings revealed a perforation of the esophagus. The death of this animal was not treatment related. There were no treatment-related effects seen in the clinical signs, group mean body weights, food consumption, hematology parameters, organ weights, or gross or histopathologic findings. The absolute and relative kidney weights were decreased significantly in high dose males, but the biological significance of this finding (in the absence of histopathologic changes in that tissue) is uncertain. Slight (less than 2-fold) increases in ALT values were seen in males and females treated at 500 mg/kg/day and slight decreases were seen in total



protein, albumin, and sodium values of females treated at 500 mg/kg/day. These statistically significant changes may be related to test article administration. Statistically significant changes in hematological parameters occurred in high dose males (increased RDW and decreased Hb, HCT, MCV, MCH, and mean corpuscular hemoglobin concentration [MCHC]) and in high dose females (decreased Hb). However, these values were within a historically acceptable range for animals of this age and strain and are, therefore, of uncertain biological significance.

Based on the findings from the present study, the NOEL for TDF administered to rats via oral gavage daily for 28 consecutive days appears to be equal to or greater than 100 mg/kg/day and less than 500 mg/kg/day.


Concentrations of TFV were determined in plasma samples obtained during a 28-day repeated dose toxicity study after oral gavage of TDF (GS-4331-05; bis-POC PMPA) in male and female Sprague-Dawley rats (Tabulated Summary 2.6.5.4.HH, 96-TOX-4331-003-PK). Blood samples (n = 2/time point) were obtained on Days 1 and 28. Time points included predose, 0.25, 0.5, 1, 2, 4, 8, and 24 hours postdose. Samples were processed for plasma and concentrations of TFV in plasma were determined using a validated reverse-phase ion-pair HPLC method with fluorescence derivatization. Plasma concentration values were subjected to pharmacokinetic analysis using noncompartmental methods. Following the first oral dose in male rats, concentrations of TFV in plasma reached mean peak values of 0.207, 0.857, and 1.13 g/mL at the 20, 100, and 500 mg/kg/day dose levels, respectively. For female rats, the Cmax following the first dose of 500 mg/kg/day was 1.91 g/mL. The Tmax in plasma ranged between 0.25 to 1 hour. Due to insufficient plasma data obtained through 24 hours for the 20- and 100-mg/kg/day dose cohorts on Days 1 and 28, complete pharmacokinetic analysis was only determined for the 500-mg/kg/day dose cohorts. AUCinf values on Day 1 for the 500 mg/kg/day dose group were 25.6 and 12.5 g·h/mL for females and males, respectively. This apparent difference in clearance between sexes is confounded by the large percentage of extrapolated AUC (48%–65%). On Day 28, the AUC0-t values for the 500 mg/kg/day dose group were 13.7 and 17.0 g·h/mL for females and males, respectively. Following oral administration of TDF on Day 1, Cmax deviated from dose proportionality over the range of 20 to 500 mg/kg/day, indicating decreased absorption at the higher dose. Repeated administration of TDF over a 28-day period resulted in only minor changes in pharmacokinetics.

2.6.6.3.2.8. Tenofovir DF: A 13 and 42-Week Oral Gavage Toxicity Study (With a 13-Week Recovery Period) of bis-POC PMPA (GS-4331-05) in the Albino Rat (97-TOX-4331-002)

The toxicity of TDF (bis-POC PMPA; GS-4331-05) in Sprague-Dawley rats was evaluated following oral gavage administration once daily for 13 or 42 consecutive weeks at doses of 0 (vehicle), 30, 100, 300, or 1000 mg/kg/day (Groups 1–5 respectively, 20/sex/group; Tabulated Summary 2.6.7.7.L, 97-TOX-4331-002). Additional animals (5/sex) in the control and treated groups were left untreated for a 13-week drug free recovery period. Cohorts of



8/sex in each of the test article-treated groups were included for toxicokinetic blood sample analysis only on Day 1 and at Weeks 13, 26, and 42. Toxicity associated with test article administration was evaluated by monitoring clinical signs of toxicity (BID), detailed physical examinations (weekly), body weights (weekly), and food consumption (weekly); ophthalmology at Weeks 13, 26, 42, and 55; hematology, clinical chemistry, and urinalysis values at Weeks 13, 26, 42, 48, and 55; markers of bone turnover from urine (Weeks 12, 25, 41, 47, and 54) and serum (Weeks 14, 43, 49, and 56); gross necropsy findings, organ weight changes, and histopathologic effects in protocol-selected tissues and bone densitometry (Weeks 14, 43, and 56).

No test article-related mortalities occurred in this study. Administration of bis-POC PMPA resulted in a dose-dependent increase in the incidence and severity of salivation in animals in all treated groups. Suppression of body weight gain was significant in males (300 and 1000 mg/kg/day), with a trend in females (1000 mg/kg/day). Decreases in mean food consumption were observed in males (1000 mg/kg/day). By the end of the recovery period (Week 56), the body weight gains and food consumption were comparable to controls; however, the body weights of the 1000-mg/kg/day dose group remained lower than controls. No test article-related ocular findings were observed. In the 1000-mg/kg/day dose group (males and females), statistically significant changes in hematology parameters over the course of the study included increased WBC and segmented neutrophil counts; increased RBC counts; decreased HCT and related red cell parameters (Hb, MCV, MCH, and/or MCHC); increased RDW; and increased platelet counts and mean platelet volume (females). No consistent changes in hematology parameters were seen in dose groups treated with 300 mg/kg/day or less. No significant changes in hematology parameters were observed in the recovery animals. Changes in serum chemistry parameters included dose-related, slight to moderate decreases in cholesterol in males ( 100 mg/kg/day); moderate to marked decreases in triglyceride levels in males and females (300 and/or 1000 mg/kg/day); slight increases ( 2-fold) in ALT activity in males ( 100 mg/kg/day) and females (1000 mg/kg/day); slight increases in AST activity (1000 mg/kg/day); marginal to slight increases in phosphorus (1000 mg/kg/day); slight decreases in total protein and globulin levels (1000 mg/kg/day) and marginal to slight increases in albumin/globulin (A/G) ratios in males (300 and 1000 mg/kg/day); slight to moderate decreases in serum bicarbonate in females ( 300 mg/kg/day) (analyzed only at Week 42); and a marginal increase in creatinine in males and females (1000 mg/kg/day). No changes in serum chemistry parameters were observed at the end of the recovery period. Urinalysis showed slight decreases in urinary pH and increases in amorphous urate crystals in males and females in the 1000-mg/kg/day dose group during the treatment period.



An evaluation of toxicity to bone was added to the protocol after study initiation (Week 12/13) and included the assessment of biochemical markers of bone turnover and bone densitometry. The mean urinary deoxypyridinoline was increased at various sampling times in males and/or females ( 100 mg/kg/day); it was comparable to controls during the recovery period. Serum osteocalcin was increased at variable sampling times in males ( 300 mg/kg/day) and/or females (1000 mg/kg/day) during the treatment period; it remained increased during the recovery period in males in the 1000 mg/kg/day group. Urinary calcium and phosphorus levels (normalized for creatinine) were increased at every sampling time (Weeks 12, 25, and/or 41) in males and/or females (1000 mg/kg/day), and approximated control values at recovery. Serum parathyroid hormone (PTH) values were notably variable; however, it was marginally increased in the 300 and 1000 mg/kg/day groups during the treatment and recovery periods.

No gross or histopathologic changes were observed in the bone. Peripheral quantitative computed tomography (pQCT) was performed on the distal femur metaphysis and mid-femur diaphysis. There were decreases in bone mineral content and bone mineral density of the total slice and trabecular and cortical/subcortical areas for males and females (1000 mg/kg/day) after 13 or 42 weeks of treatment; consistent with increased bone resorption. Decreases in cortical thickness generally correlated with slight decreases in periosteal circumference and slight increases in endosteal circumference. Similar effects on the cortex were observed at the mid-femur diaphysis with evidence of a similar dose-related effect for males (300 mg/kg/day) at Week 43. There was evidence for reversal of bone changes following a 13-week recovery period, most notably at the femoral metaphysis. There were no test article-related effects on bone parameters at the 30- or 100-mg/kg/day dose levels.



At necropsy, absolute and or relative adrenal weights were increased in the 1000-mg/kg/day dose group. In the absence of any associated histopathologic changes, the toxicological relevance of these findings is uncertain. Gross findings considered to be test article related at Week 14 and/or Week 43 necropsies included thickening of the wall in the duodenum and jejunum and depressed stomach areas, with incidence related to dose and duration of treatment (primarily in the 1000-mg/kg/day group). Test article-related histopathologic findings were observed in the kidneys and GI tract. Slight to mild renal tubular karyomegaly occurred in the 300 (males) and 1000 mg/kg/day groups at Week 14 and in all test article groups at Week 43. It was still present after a 13-week recovery in the 300 (males) and 1000 mg/kg/day dose groups. A dose-related increase in the incidence and severity (slight to moderate) of renal tubular pigment was observed at Week 43 in all dose groups. Slight renal tubular pigment occurred in 1 rat in each of the 100-, 300-, and 1000-mg/kg/day dose groups at Week 56. Gastrointestinal changes included slight to moderate typhlitis (males) and gastritis ( 300 mg/kg/day at Weeks 14 and 43) and slight to mild duodenal epithelial cell hypertrophy at Week 14 (1000 mg/kg/day) and Week 43 ( 100 mg/kg/day). At Week 43, slight to mild colitis (males); slight to moderate mucosal hyperplasia of the duodenum at

300 mg/kg/day; slight to moderate jejunitis, ileitis, duodenitis, and epithelial cell hypertrophy of jejunum; slight villous atrophy of ileum; and slight to moderate mucosal atrophy of cecum were observed in males (1000 mg/kg/day). Slight to moderate mucosal atrophy of colon was seen in males and females (1000 mg/kg/day) at Week 43. In the recovery animals, GI effects were generally reversed. A trend for reversal of renal changes was noted based on decreased incidence and/or severity of renal tubular karyomegaly and tubular pigment accumulation.

As test article-related renal and/or GI pathology was observed at all dose levels evaluated in this study, a NOAEL was not clearly established. The reduced incidence and/or severity of renal and GI pathology following a 13-week recovery period suggest that toxicity is reversible.




Concentrations of TFV were determined in plasma samples obtained during the 42-week repeated dose toxicity study (Tabulated Summary 2.6.5.4.DD, 97-TOX-4331-002-PK). Plasma samples were taken from rats (2 animals/sex/time point) at Day 1, Week 13, Week 26, and Week 42 following daily oral dosing of 30, 100, 300, or 1000 mg/kg/day of TDF. Pharmacokinetic parameters for TFV in plasma were determined by noncompartmental methods. No apparent differences were observed in the pharmacokinetics of TFV between male and female rats at any dose or time point throughout the study. On Day 1, mean Cmax ( g/mL) values were 0.46, 1.17, 1.51, and 2.71 for the 30-, 100-, 300-, and 1000-mg/kg/day dose cohorts, respectively. The corresponding calculated mean AUCinf were 2.87, 6.64, 17.70, and 43.50 g·h/mL. At Day 1, there was a better correlation between calculated AUC values and dose than between plasma Cmax and dose, suggesting that higher doses were more slowly absorbed. Compared to Day 1, mean plasma AUC0 24h at the 30-, 100-, and 1000-mg/kg/day doses were increased by 32%, 25%, and 49%, respectively following 42 weeks of daily dosing. At the 300-mg/kg/day dose, no change in AUC was observed following 42 weeks of dosing. Therefore, no definitive dose-dependent changes were observed in the pharmacokinetics of TFV following 42 weeks of daily oral dosing of TDF.

2.6.6.3.2.9. Emtricitabine/Tenofovir DF: A 14-Day Oral Gavage Toxicity Study Comparing Non-Degraded and Degraded TDF/FTC in Sprague-Dawley Rats (TX-164-2001)

The purpose of this study was to investigate the potential toxicity of nondegraded and degraded TDF/FTC following daily oral administration to the rat for a minimum of 14 days (Tabulated Summary 2.6.7.7.M, TX-164-2001). Animals (10 males/group) were administered doses of 30/20, 100/67, or 300/200 mg/kg/day of nondegraded or degraded TDF/TFC in suspension vehicle at a dose volume of 10 mL/kg. The following parameters were measured during the study: mortality, clinical signs, detailed physical examination, body weights, food consumption, laboratory investigations (hematology, biochemistry, and urinalysis), gross pathology, organ weight analysis, and histopathology.

In this study there were no deaths related to treatment with TDF/FTC (degraded or nondegraded). Clinical signs were limited to dose-related postdosing salivation. There were no effects on body weight or food consumption considered to be related to treatment with TDF/FTC (nondegraded and degraded). There were no toxicologically significant treatment-related changes in hematology, biochemistry, or urine parameters. Organ weight evaluation revealed marginal increases in the weights of adrenal glands (noted in all treated groups but those receiving degraded TDF/FTC at the lowest dose). No gross or histological changes were identified which might account for this increased adrenal weight. No treatment-related gross changes were observed at necropsy. Microscopic evaluation revealed hyperplasia of the anterior duodenal mucosa overlying Brunner’s glands. It was seen in 7/10 animals treated at the high-dose level with nondegraded TDF/FTC and in 2/10 animals receiving degraded TDF/FTC and was considered to be treatment related.



Daily oral administration of nondegraded and degraded TDF/FTC at dose levels of 30/20, 100/67, and 300/200 mg/kg/day for 14 consecutive days to male Sprague-Dawley rats was well tolerated. There were no toxicologically significant differences between groups treated with nondegraded and degraded TDF/FTC. A minor increase in adrenal weights at necropsy (all dose levels, except those receiving degraded TDF/FTC at the lowest dose) and a hyperplasia of the anterior duodenal mucosa overlying Brunner’s glands was seen in animals treated at the high-dose level with nondegraded and degraded TDF/FTC.

In conclusion, the NOAEL in this study was considered to be 100/67 mg/kg/day.

2.6.6.3.3. Rabbit

2.6.6.3.3.1. Rilpivirine: 5-Day Oral Dose Range Finding Toxicity Study with TMC278 Base in Rabbits

This nonpivotal study was part of the dose range finding studies for the embryo-fetal development study with TMC278 base in rabbits. For studies with pregnant rabbits, see Section 2.6.6.6.2.6.

TMC278 base suspended in 0.5% (m/v) aqueous HPMC was administered by oral gavage for 5 days to New Zealand white rabbits (Tabulated Summary 2.6.7.6.A, TMC278-NC126). Groups of 5 nonpregnant females received 0 (vehicle), 100, 300, or 1000 mg/kg in 10 mL/kg. Daily observations were made for clinical signs, morbidity, and mortality. Body weight and food consumption were determined at the end of the dosing period. Blood samples for hematology, serum chemistry, and toxicokinetic analyses were taken at the end of the dosing period. At necropsy, gross lesions of external features and body cavities were recorded, but no organs were weighed and no organs or tissues were taken for histopathology.

No mortalities occurred. Food consumption was strongly reduced in a dose-related fashion. The animals dosed with 100 mg/kg/day consumed less than 75% of the food and those given 1000 mg/kg/day less than 10% of the food consumed by the control animals. Consequently, fecal output and body weight were significantly reduced. Mean reticulocyte counts were strongly and dose-dependently reduced and mean creatinine levels were increased in animals dosed with 300 and 1000 mg/kg/day. No relevant changes were noted at necropsy. Toxicokinetics showed mean Cmax values of 57.3, 124, and 138 μg/mL and mean AUC0 24h

values of 1120, 2695, and 2971 g.h/mL in groups dosed with 100, 300, and 1000 mg/kg/day, respectively.

2.6.6.3.4. Dog

2.6.6.3.4.1. Rilpivirine: Non-Pivotal Pilot Oral Toxicity with TMC278 Base and TMC278 – TMC278 Bridging Studies

The pivotal studies with administration durations ranging from 1 to 12 months were preceded by pilot studies. Following the selection of the HCl salt for further final development, a 1-month bridging study with TMC278 base and TMC278 was done.



5-Day Pilot Oral Toxicity Study

TMC278 base dissolved in PEG400 + CA was administered once daily, by oral gavage, for 5 days to groups of beagle dogs each comprising 1 male and 1 female at 0 (vehicle) and 80 mg/kg/day in 0.8 mL/kg, after a wash-out period of 7 days following a similar single dose (Tabulated Summary 2.6.7.5.B, TMC278-Exp5461; Tabulated Summary 2.6.7.2.B, FK4102 [TOX5461]).

No test article-related effects were noted on clinical signs, body weight, clinical pathology, and postmortem parameters. Exposure data on Day 5 showed a Cmax value for the male of 13 μg/mL and for the female of 8.0 μg/mL. The respective AUC0 24h values were 262 and 154 μg.h/mL.

7-Day Pilot Oral Toxicity Study

TMC278 base dissolved in PEG400 + CA was administered once daily, by oral gavage, for 7 days to groups of 4 male beagle dogs at 0 (negative control, water), 0 (vehicle), 20, 40, or 80 mg/kg/day in 1 mL/kg (Tabulated Summary 2.6.7.6.A, TMC278-TOX5534; Tabulated Summary 2.6.7.2.B, FK4244 [TOX5534]).

There were no mortalities. Serum levels of total bilirubin (111% and 108% at 40 and 80 mg/kg/day, respectively), and glucose and cholesterol (11% and 10%, respectively, both at 80 mg/kg/day) were increased. The AUCs of cortisol concentrations in serum were decreased 56% to 75% in all groups treated with TMC278. Multifocal inflammatory cell infiltration and multifocal vacuolated cells in the zona fasciculata of the adrenal glands were noted in all treated groups. Systemic exposure expressed as Cmax and AUC0 24h values are given in Table 12 below.

Table 12. Exposure to TMC278 (Cmax and AUC) in a 7-Day Oral Toxicity Study with TMC278 Base in Dogs

Cmax (μg/Ml) AUC0-24h (μg.h/Ml) Dose(mg/kg/day) Sampling Days M (n = 4)

1 1.6 18 20

7 3.7 39

1 1.7 27 40

7 7.7 159

1 1.5 19 80

7 7.8 147

M: males



1-Month Bridging Study with TMC278 Base and TMC278 in Dogs

This study served to bridge the toxicity and toxicokinetics of TMC278 base dissolved in PEG400 + CA with those of TMC278 suspended in 0.5% (m/v) aqueous HPMC. Both forms were administered once daily by oral gavage to groups of 3 male and 3 female beagle dogs for 1 month at 0 (vehicle), 5, or 40 mg/kg/day in 1 mL/kg (Tabulated Summary 2.6.7.6.A, TMC278-NC116).

No mortalities occurred in this study. There were no significant differences in the toxicity parameters. Effects were almost exclusively observed in the groups dosed with both forms at 40 mg/kg/day.

Reduced RBC (7%, females) and reticulocyte (up to 61%, males) counts, and HCT (15%) were noted.

Increased serum concentrations of bilirubin (up to 2-fold) and increased activities of ALT (up to 6-fold), AST (14%, females), ALP (up to 2-fold), and gamma-glutamyltransferase (GGT; up to 6-fold) were recorded. Moreover, serum concentration of triglycerides (up to 53%) and inorganic phosphate (up to 11%) were decreased.

Endocrinology showed increased serum concentrations of ACTH (92%), progesterone (up to 11-fold, males), and 17 -hydroxyprogesterone (also in males given 5 mg/kg/day; up to 34-fold in males dosed with 40 mg/kg/day); and decreased levels of cortisol (up to 54%).

At necropsy, increased weights of liver (up to 19%) and adrenal glands (up to 32%) were recorded. Histopathology lesions comprised swollen cells with densely stained cytoplasm in zona reticularis/fasciculata and presence of foamy cells in the zona fasciculata of adrenal glands. Moreover, an increase in the number of cystic luteinized follicles or tertiary follicles in the ovaries was noted, with a tendency towards an increase in glandular development in the uterus. In testes, prominent Leydig cells were noted in 1 animal. Males and females showed centrilobular/periportal inflammation, brown pigmented macrophages, single cell death, and oval cell proliferation in liver.

Systemic exposure expressed as Cmax and AUC0 24h values are given in Table 13 below.



Table 13. Exposure to TMC278 (Cmax and AUC) in a 1-Month Oral Bridging Study with TMC278 Base and TMC278 in Dogs

Cmax (μg/Ml) AUC0 24h (μg.h/Ml) TMC278 Base (mg/kg/day) Sampling Days M (n = 3) F (n = 3) M (n = 3) F (n = 3)

1 1.0 0.66 12 8.1 5

28 1.4 1.1 23 14

1 1.0 3.5 17 59 40

28 3.8 2.5 73 43

TMC278 (mg/kg/day)

1 0.49 0.53 6.9 6.2 5

28 0.89 0.70 13 7.5

1 1.3 1.5 20 22 40

28 4.1 5.8 76 81


2.6.6.3.4.2. Rilpivirine: 1-Month Toxicity Study Including 1-Month Recovery in Dogs

TMC278 base formulated in PEG400 + CA was administered once daily, by oral gavage, for 1 month to groups of 3 male and 3 female beagle dogs at 0 (water, negative control), 0 (vehicle), 5, 10, or 40 mg/kg/day in 1 mL/kg (Tabulated Summary 2.6.7.7.N, TMC278-Exp5650). To evaluate reversibility of the effects of TMC278 in a 1-month postdosing period, 2 males and 2 females were added to the negative control group and the group treated with 40 mg/kg/day. Regular observations were made for clinical signs, morbidity, and mortality. Body weight and food consumption were recorded weekly. Ophthalmic examinations and electrocardiogram (ECG) recordings were performed and samples for hematology, serum chemistry, and urinalysis were taken prior to dosing and at the end of the 1-month dosing and postdosing periods. Serum concentrations of progesterone, cortisol, aldosterone, ACTH, and estradiol (females, only) were determined in samples taken on Days 1 and 16, and towards the end of the dosing and postdosing periods. For the determination of AUCs for ACTH, cortisol, and aldosterone, 4 samples were taken over a 24-hour period. Progesterone and estradiol were determined in predosing and 4-hour samples. Blood samples for toxicokinetics were taken on Day 1 and towards the end of the dosing and postdosing periods. Single blood samples for trough level and elimination determinations were taken regularly during the dosing and recovery periods. At necropsy, blood and adrenal gland samples were taken for determination of TMC278. Moreover, gross lesions and organ weight were recorded and tissues and organs were sampled for histopathology. Adrenal gland samples of the dogs of the negative and vehicle groups, and the group dosed with 40 mg/kg/day, were examined by electron microscopy.



No mortalities occurred in this study. Red vaginal discharge was noted in 2 out of 3 females dosed with 10 mg/kg/day and 1 out of 5 dosed with 40 mg/kg/day. Soft feces and vomiting occurred in all groups except the negative control group indicating this effect was vehicle related rather than test article related. There were no relevant effects on ophthalmoscopy, heart rate, ECGs, or hematology. Male dogs dosed with 40 mg/kg/day lost body weight during the first 3 weeks of dosing. Female dogs in this group did not gain body weight throughout the dosing period. These effects were associated with a reduced food intake.

Red blood cell count, Hb, and HCT of samples from males and females treated with 40 mg/kg/day taken together were marginally (less than 5%) lower than values from the vehicle group. White blood cell count in this group was 12% higher than that of the vehicle group. Albumin and total protein concentrations in samples of males and females taken together in the groups dosed with 10 and 40 mg/kg/day were marginally lower than those of the vehicle group, whereas triglyceride concentrations were 14% and 21% lower, respectively. The concentrations of cholesterol (32%) and total bilirubin (136%) of animals dosed with 40 mg/kg/day were higher than those of the vehicle group. In the same group, the activities of ALP and ALT were respectively 120% and almost 3-fold higher than those of the vehicle group.

Progesterone concentrations were increased in a more or less dose-related fashion in the trough level samples, more pronounced in the samples taken 4 hours after dosing on Day 16, and even more towards the end of the dosing period in males and females treated with TMC278. The increases were almost 30- and 10-fold in males and females, respectively, dosed with 40 mg/kg/day. The AUCs of ACTH were increased at the end of the dosing period, without a clear dose relationship or statistical significance. The AUCs of cortisol showed a tendency for decrease in the animals dosed with 10 and 40 mg/kg/day. After a 1-month postdosing period, all parameters showed similar values in the control group and the group treated with 40 mg/kg/day.

Mean absolute weight of ovaries showed a more or less dose-related increase ranging from 33% in the group dosed with 5 mg/kg/day up to 160% and 138% in the groups given 10 and 40 mg/kg/day, respectively, compared to the vehicle group. At necropsy, a swollen aspect of the female genital tract comprising cervix, ovaries, uterus, and vagina and of the mammary glands was noted in the majority of the animals dosed with 10 and 40 mg/kg/day.

In the adrenal cortex, histopathology showed an increase in the number of swollen cells with dense cytoplasm and reduced Oil red O-staining indicative of a reduction of neutral fat content in the animals dosed with 10 and 40 mg/kg/day compared with the vehicle group. The ultrastructural evaluation of adrenal gland samples from males and females treated with 40 mg/kg/day showed a reduction in the number of cytoplasmic lipid vacuoles in endocrine cells. This explains and confirms the light microscopy observations of less Oil red O-staining and dense cytoplasm. In addition to these findings, an increase of macrophage-like cells containing many small- and large-sized lipid vacuoles and secondary lysosomes with lipid and lamellar material was noted in the zona fasciculata. The higher number of these cells containing material representing membrane detritus indicates that TMC278 may have a mild degenerative effect in this zone.



The female genital tract showed increased activation in the groups treated with 10 and 40 mg/kg/day. In the ovaries, corpora lutea were detected in 2 animals dosed with 10 mg/kg/day and in 1 animal that received 40 mg/kg/day. This finding was associated with prominent high epithelium in the oviduct; increased glandular development in the uterus; cornification with increased thickness of the epithelium of the cervix and vagina; and with active alveolar development in mammary glands. More prominent tertiary follicles were noted in all test article-treated animals that had not ovulated at the end of dosing period.

In liver, minimal to moderate centrilobular perivascular inflammatory reaction was observed in males. Minimal increase in the number of multifocally dispersed centrilobular hepatocytes with a clear appearance was noted in the groups treated with 10 and 40 mg/kg/day. Moreover, a slight to moderate increase of MPS-aggregates; slight to minimal centrilobular hepatocellular single cell necrosis and multifocal centrilobular perivascular fibrosis; and minimal multifocal bile duct proliferation occurred in males and females dosed with 10 and 40 mg/kg/day.

All adverse effects that were seen at 40 mg/kg/day were completely reversible within a 1-month recovery period, except for the changes in the liver and the increased level of ALP in the serum. However, the histological changes in the liver showed signs of reversibility.

Given the dose-related trend in endocrinology results and ovarian weight already evident in the group treated with the low dose, 5 mg/kg/day, a NOEL was not established. Systemic exposure expressed as Cmax and AUC0 24h values are given in Table 14 below. The mean adrenal gland to plasma ratios of the concentrations of TMC278 ranged from 1.3 to 3.6, without a clear dose relationship. After 1 month of recovery, the concentration of TMC278 in adrenal gland, as well as in plasma, was below the limit of quantification.

Table 14. Exposure to TMC278 (Cmax and AUC) in a 1-Month Toxicity Study Including 1-Month Recovery with TMC278 Base in Dogs

Cmax (μg/Ml) AUC0 24h (μg.h/Ml) Dose(mg/kg/day) Sampling Days M (n = 3) F (n = 3) M (n = 3) F (n = 3)

1 0.94 1.3 13 15 5

29 1.5 2.0 27 37

1 1.3 1.3 22 14 10

29 5.6 2.5 103 47

1 2.8 2.4 51 40 40

29 12 9.5 204 160




2.6.6.3.4.3. Rilpivirine: 6-Month Toxicity Study Including 3-Month Interim Evaluation with TMC278

TMC278 base formulated in PEG400 + CA was administered once daily, by oral gavage, for 6 months to groups of 6 male and 6 female beagle dogs at 0 (vehicle), 5, 10, or 40 mg/kg in 1 mL/kg (Tabulated Summary 2.6.7.7.O, TMC278-NC115). Regular observations were made for clinical signs, morbidity, and mortality. Body weight and food consumption were recorded weekly. Ophthalmic examinations, ECG recordings, and samples for clinical pathology were taken prior to dosing, prior to 3-month interim evaluation, and at the end of the dosing period. Clinical pathology comprised a standard set of hematological, serum chemistry, and urinalysis parameters. In addition, serum concentrations of 17 -hydroxyprogesterone, progesterone, cortisol, and ACTH were determined. For the determination of AUCs for ACTH and cortisol, 4 samples were taken over a 24-hour period from the first 3 male and 3 female animals in each group. Blood samples for toxicokinetics were taken toward the 3-month interim evaluation, and towards the end of the dosing period. In addition, on the day of necropsy, just prior to the sacrifice, a single blood sample and samples of liver and adrenal gland were taken for the determination of TMC278. After 3 months of treatment, the last 2 males and 2 females of each group were killed for the interim evaluation. The remaining animals were killed at the end of the 6-month treatment period. At both necropsies, gross lesions and organ weight were recorded, and tissues and organs were sampled for histopathology.

No mortalities occurred in this study. The males and females dosed with 40 mg/kg/day lost body weight during the first 4 weeks of treatment. Thereafter, their weekly body weight gain was not different from that of the control animals. The effects on body weight are considered associated with the reduction of food consumption noted in the animals treated with 40 mg/kg/day during the first 4 weeks of treatment. There were no relevant effects on heart rate, ECG, ophthalmology, hematology, and urinalysis.

Serum chemistry towards 3 months of dosing showed 40% and 23% increases in cholesterol concentrations of males and females, respectively, dosed with 40 mg/kg/day. Total bilirubin was increased 46% in females treated with 10 mg/kg/day, and 31% and 77% in males and females, respectively, dosed with 40 mg/kg/day. Alkaline phosphatase activity was increased 85% in the females dosed with 5 and 10 mg/kg/day, and almost 3.5-fold in females dosed with 40 mg/kg/day. The increase in ALP activity was 28% and 72% in males treated with 10 and 40 mg/kg/day, respectively. The same parameters were increased at the end of the 6-month treatment period to an almost similar extent as after 3 months of treatment, except ALP. The ALP enzyme activity in males and females dosed with 40 mg/kg/day had increased 130% in males and almost 5.5-fold in females.



Cortisol precursor 17 -hydroxyprogesterone showed a more or less dose-related increase in males only, from more than 4-fold in the group treated with 5 mg/kg/day, and up to almost 50-fold after 3 months of dosing and 20-fold after 6 months of dosing with 40 mg/kg/day. The assessment of the progesterone levels in females after 3 months of treatment was hampered by high and highly variable levels in the vehicle group, which were probably associated with the phase of the estrous cycle of these animals. At the end of the 6-month dosing period, control levels in both males and females were similar and a dose-related increase in the groups treated with TMC278 base was noted, ranging from 100% in the animals dosed with 10 mg/kg/day to 4- to 5-fold in the animals dosed with 40 mg/kg/day. The AUCs of cortisol showed a dose-related decrease, which was similar after 3 and 6 months of treatment. This decrease was more prominent in males, ranging from 30% (males only) in the group dosed with 5 mg/kg/day and up to 50% to 60% in the group dosed with 40 mg/kg/day. The AUCs of ACTH showed clear increases from 80% in males dosed with 5 mg/kg/day for 6 months up to almost 3-fold in the high dose males at the end of the dosing period. Females showed similar effects on cortisol and ACTH, but to a lower extent than the males after 3 months of treatment. However, at the end of the dosing period females did not show a clear effect on these hormones.

Histopathology showed that the genital tract, adrenal glands, liver, gall bladder, and thymus of male and females were affected by TMC278. In the zona fasciculata and zona reticularis of the adrenal cortex, an increase of swollen cells with densely stained cytoplasm and a lower number of droplets stainable with Oil Red O was noted in the groups treated with 10 and 40 mg/kg/day after 3 and 6 months of treatment. At the end of the dosing period, a single male and female treated with 5 mg/kg/day also showed these effects. Thymus of 1 male and 1 female treated for 3 months with 40 mg/kg/day showed slight to marginal signs of involution. A minimal number of macrophages laden with presumably lipogenic (Perl’s negative) pigment were noted perivascularly in liver in 1 male dosed for 3 months with 40 mg/kg/day, in 2 males dosed for 6 months with 10 mg/kg/day, and in 2 males and 2 females dosed for 6 months with 40 mg/kg/day. Brown pigmentation of the gall bladder epithelium was noted to a minimal degree in 1 male dosed with 10 mg/kg/day, and in the majority of males and females dosed with 40 mg/kg/day at the end of the dosing period. In testes, Leydig cell hypertrophy occurred in 1 animal dosed with 10 mg/kg/day (minimal), in the 2 animals dosed with 40 mg/kg/day (minimal to slight) after 3 months of treatment, and in 2 animals (minimal) treated for 6 months with 40 mg/kg/day. Ovaries were increased in weight in all TMC278-treated groups after 3 months, and in the animals that received 10 and 40 mg/kg/day (100%) after 6 months of dosing. In the majority of the animals treated with 40 mg/kg/day for 6 months, ovaries, uterus, and vagina had a swollen aspect. Histopathology showed a slight increase in the number of atretic follicles of animals treated with 10 and 40 mg/kg/day for 3 and 6 months. In animals treated with 40 mg/kg/day, the number of regressive corpora lutea was slightly increased at the 3- and 6-month autopsies. The number of tertiary follicles was increased in animals of all groups treated with TMC278 for 3 and 6 months.



Given the changes seen in adrenals and ovaries at the lowest dose of 5 mg/kg/day, no NOAEL was established. Systemic exposure expressed as Cmax and AUC0 24h values are given in Table 15 below. The mean adrenal gland to plasma ratios of the concentrations of TMC278 ranged from 4.1 to 8.1 over the groups, without a clear dose relationship. Liver to plasma ratios of the concentrations of TMC278 ranged from 7.7 to 13. Adrenal and liver to plasma ratios did not change between Day 93 and Day 184/185 of treatment.

Table 15. Exposure to TMC278 (Cmax and AUC) in a 6-Month Toxicity Study with TMC278 Base in Dogs


1 1.0 0.70 11 9.2

86 1.4 1.1 21 18 5

177 1.5 1.4 21 17

1 1.3 1.2 22 20

86 2.0 2.2 28 27 10

177 2.0 1.9 26 32

1 1.5 0.82 23 10

86 2.5 3.1 41 52 40

177 4.0 2.9 68 43


2.6.6.3.4.4. Rilpivirine: 12-Month Toxicity Study with TMC278

TMC278 base formulated in PEG400 + CA was administered once daily, by oral gavage, for 12 months to groups of 4 male and 4 female beagle dogs at 0 (vehicle), 5, 10, or 40 mg/kg in 1 mL/kg (Tabulated Summary 2.6.7.7.P, TMC278-NC107). Regular observations were made of clinical signs, morbidity, and mortality. Body weight and food consumption were recorded weekly. Ophthalmic examinations and ECG recordings were made and samples for hematological, serum chemistry, and urinalysis were taken prior to dosing; after 1 week (ECGs only); after 1, 3, 6, and 9 months of treatment; and at the end of the dosing period. Serum concentrations of 17 -hydroxyprogesterone, progesterone, estradiol, testosterone (in males only), and luteinizing hormone (LH, in males only) were determined in samples taken prior to the first administration (no samples for testosterone and LH); prior to dosing after 3 (no samples for testosterone and LH), 6, and 9 months of treatment; and at the end of the treatment period. For the determination of AUCs for ACTH and cortisol, 4 samples were taken over a 24-hour period before the first administration and after 3, 6, 9, and 12 months of treatment at 1, 2, 4, and 24 hours after a daily dose of TMC278. Blood samples for toxicokinetics were taken on Day 1, after 3 and 6 months of treatment, and prior to necropsy. At necropsy, gross lesions and organ weight were recorded, and tissues and organs were sampled for histopathology. The female animal dosed with 10 mg/kg/day that was killed for humane reasons following a gavage error on Day 21 was replaced.



No test article-related mortalities occurred. Five animals, including 1 control male (Day 360), 2 males (Days 155 and 333) and 1 female (Day 20, replaced) dosed with 10 mg/kg/day, and 1 female (Day 54) treated with 40 mg/kg/day, were killed for humane reasons in a moribund condition. Postmortem evaluations indicated that their condition was caused by respiratory tract abnormalities probably due to the presence of the applied formulation.

Body weight gain in males at the end of the study was reduced in a dose-related fashion in all groups (40% reduction in males dosed with 40 mg/kg/day). In females, a dose-dependent reduction of body weight gain occurred in groups treated with 10 and 40 mg/kg/day. At 40 mg/kg/day, the reduction was more than 50%. Food consumption in males was hardly affected by treatment. However, in females, food consumption varied considerably throughout the treatment period and was frequently reduced in the animals dosed with 10 and 40 mg/kg/day.

No test article-related abnormalities were detected at ophthalmic examinations or on ECGs. Hematology, serum chemistry, and urinalysis parameters were affected only in samples taken at the end of the dosing period from animals treated with 40 mg/kg/day. Red blood cell count, Hb, and HCT in males were 11% to 13% lower. Serum calcium concentrations in males and females were decreased 10% and 7%, respectively. Inorganic phosphate concentration in females was increased 17%. Creatinine level in males was 14% higher than the control value. Total bilirubin concentrations were 15% and 42% higher in males and females, respectively. Alkaline phosphatase activity in males was almost twice as high as in controls, whereas the enzyme activity in females was 55% higher. Urinary volume in males was 3.5-fold higher.

Hormone analyses showed a dose-related increase ranging from 3- to 20-fold in the serum concentrations of both progesterone and 17 -hydroxyprogesterone in males in all groups treated with TMC278 base throughout the dosing period. Evaluation of these hormones in females was hampered by interference of the estrous cycle, but the serum concentrations showed an increased trend similar to what was evident in males. The AUCs for cortisol in males showed a considerable variation, but for females a dose-related decrease ranging from minimally 14% to 80% was determined over all test article-treated groups, which was statistically significant in females dosed with 10 and 40 mg/kg/day throughout the dosing period. The AUCs for ACTH showed no effects, except for an incidental increase in the group treated with 40 mg/kg/day. At the end of the treatment period, LH and testosterone levels in males showed no treatment-related effects. Estradiol was essentially undetectable in the males of all groups throughout the study. In females, the concentrations of this hormone were highly variable due to the estrous cycle, but did not show any trend of an effect and were consistent with progesterone levels on an individual basis.



Terminal body weight showed a dose-related reduction in males, notably in all groups treated with TMC278. Mean adrenal gland weight was increased in animals treated with 40 mg/kg/day. The absolute increase was 23% in both males and females. The increase relative to body weight was 50% in males and 46% in females. This increased organ weight is probably associated with the (slight to moderate) increase in the size of the zona fasciculata in the adrenal cortex of 1 male and 3 females treated with 40 mg/kg/day and the bilateral focal hypertrophy of cells in this zone in 1 male of the same group. Moreover, histopathology showed dense cytoplasm of the cells in the zona fasciculata and/or reticularis of all females treated with TMC278, including the premature death animals; a single male dosed with 10 mg/kg/day; and the majority of the males treated with 40 mg/kg/day. This effect was usually associated with a minimal reduction of cellular neutral fat deposits. In the high dose group, half of the males and females showed pigment deposits in the adrenal cortex.

The weight of ovaries showed a dose-related increase in females dosed with 10 and 40 mg/kg/day. The absolute increase was respectively 40% and 120% of control values. The increase relative to body weight was respectively 48% and 170% of control values. These weight effects are likely associated with the increase in the number of antral follicles and the prominent corpora lutea in the animals dosed with 10 and 40 mg/kg/day. In liver, yellow pigmentation in hepatocytes and canaliculi was noted in all males and females given 40 mg/kg/day and in 1 female given 10 mg/kg/day. Prominent brown pigment in the epithelium of the gall bladder was noted in males given 40 mg/kg/day. In testes, minimal hypertrophy of the Leydig cells was recorded in 2 males given 40 mg/kg/day. However, this effect had no impact on Sertoli cell functioning or spermatogenesis as an evaluation of the main stages of the spermatogenic cycles did not indicate any changes. Kidney changes were only seen in the group treated with 40 mg/kg/day and included acute interstitial nephritis in 2 males and minimal to slight corticomedullary mineralization in all terminally killed females.

As a consequence of the adrenal changes at the low dose group, no NOAEL was established. Systemic exposure expressed as Cmax and AUC0 24h values are given in Table 16 below.



Table 16. Exposure to TMC278 (Cmax and AUC) in a 12-Month Toxicity Study of TMC278 Base in Dogs


1 0.70 0.75 11 9.7

90 1.2 1.4 18 21

273 1.1 1.1 16 18 5

363 1.1 1.5 17 19

1 0.90 1.2 c 15 15c

90 2.0 2.3 29 29

273 1.3a 2.3 19a 31 10

363 1.3b 2.2 24b 36

1 2.4 2.5 37 41

90 3.6 5.5a 60 88a

273 3.2 3.5a 60 51a 40

363 4.1 5.5a 65 61a

M = males, F = females, a: n = 3, b: n = 2; c: n=5 samples of dog humanely killed on Day 21 and of replacement dog are included

2.6.6.3.4.5. Tenofovir DF: 5-Day Repeated Oral Dose Toxicity Study with GS-4331 in Dogs, (96-TOX-4331-001)

The objective of this repeated oral dose toxicity study was to determine the toxicokinetic profile and to evaluate the potential toxicity of GS-4331-02, an oral CA prodrug salt of TFV, when administered orally to male beagles (2/group) daily for 5 consecutive days at doses of 0, 9, 45, and 180 mg/kg/day (Tabulated Summary 2.6.7.6.B, 96-TOX-4331-001). At doses of

45 mg/kg/day, there were liquid feces; vomitus; low food consumption; body weight loss; mild to marked (1 dog at 180 mg/kg/day) increases in creatinine; minimal to moderate renal cortical tubular degeneration/regeneration with individual epithelial cell necrosis; moderate bone marrow hypocellularity (180 mg/kg/day only); and slight to moderate intestinal epithelial necrosis throughout the small intestine (180 mg/kg/day only). Based on the results of this study, the NOEL for GS-4331-02 administered to dogs by oral gavage for 5 consecutive days was determined to be 9 mg/kg/day.




In the toxicokinetic portion of study 96-TOX-4331-001, TFV concentrations were assayed from plasma samples drawn on the fifth day of drug administration by HPLC following fluorescence derivatization (Tabulated Summary 2.6.5.4.FF, 96-TOX-4331-001-PK). Overall, analysis of TFV plasma concentration data from this study revealed rapid absorption followed by biexponential elimination.

In animals dosed at 9 mg/kg/day, Cmax ranged from 0.943 to 1.54 g/mL and AUCtau ranged from 4.19 to 4.98 g·h/mL. At 45 mg/kg/day, Cmax values ranged from 6.16 to 7.31 g/mL and AUCtau ranged from 30.5 to 62.9 g·h/mL. Pharmacokinetic values for the 2 animals dosed at 180 mg/kg/day varied greatly. For the first animal, high Cmax (79.4 g/mL) and AUCtau (1840 g·h/mL) values were consistent with the toxicity finding of renal failure in this animal. In contrast, a second animal had a calculated Cmax of 11.5 g/mL and an AUCtau value of 89.1 g·h/mL.

All animals in dose groups that received GS-4331-02 demonstrated exposure based on the measurable levels of TFV in plasma on Day 5. Pharmacokinetic data and plasma profiles from this study demonstrated increased exposure of TFV, with increasing dosage of GS-4331-02 in beagle dogs.

2.6.6.3.4.6. Tenofovir DF: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 in the Beagle Dog (96-TOX-4331-004)

The objective of this study was to investigate the potential toxicity of TDF (GS-4331-05) in beagle dogs (4/sex/group) dosed via oral gavage once daily for 28 consecutive days at 0 (vehicle), 3, 10, or 30 mg/kg/day (Groups 1 4, respectively) (Tabulated Summary 2.6.7.7.Q,96-TOX-4331-004). Blood samples were taken on study Days 1 and 28 to assess systemic drug exposure. Toxicity associated with test article administration was evaluated by monitoring clinical signs of toxicity (BID); body weight (weekly); food consumption (daily); ophthalmology (Week 4); clinical chemistry, hematology, and urinalysis values (Week 4); gross necropsy findings, organ weight changes, and histopathologic effects in protocol-selected tissues.



There were no deaths or treatment-related clinical signs of toxicity noted, and no alterations of the urinalysis parameters. Treatment-related decreases in body weight were seen in males treated at 30 mg/kg/day and in food consumption of males and females treated at 30 mg/kg/day. There were slight dose-related decreases in RBC counts, Hb concentration, and HCT in males and females treated at 10 and 30 mg/kg/day, and in MCHC of females treated at 30 mg/kg/day. There were dose-related increases in total bilirubin in females treated at 10 mg/kg/day, and in males and females treated at 30 mg/kg/day. Blood urea nitrogen (BUN) and creatinine concentrations were raised in males treated at 30 mg/kg/day. There was a dose-related decrease in cholesterol concentration in males and females treated at 10 mg/kg/day and 30 mg/kg/day. Dose-related histopathologic changes associated with TDF administration were present only in kidney tissue of males and females treated at 10 or 30 mg/kg/day. Renal tubular degeneration and regeneration and renal tubular cell karyomegaly were present in all males and females dosed at 10 mg/kg/day (slight severity) and in all males and females treated at 30 mg/kg/day (mild-to-moderate severity). No test article-related histopathologic lesions were observed in animals treated at 3 mg/kg/day. Based on the findings from the present study, the NOEL for TDF administered to dogs via oral gavage daily for 28 consecutive days is equal to or greater than 3 mg/kg/day and less than 10 mg/kg/day.


Concentrations of TFV were determined in plasma samples obtained during this 28-day repeated dose toxicity study (Tabulated Summary 2.6.5.4.GG, 96-TOX-4331-004-PK). Blood samples were obtained prior to TDF dosing; at 15 and 30 minutes; and at 1, 2, 4, 8, and 24 hours postdose on Days 1 and 28. Plasma samples were assayed for TFV using a validated reverse-phase ion-pair HPLC method with fluorescence derivatization. Plasma TFV concentration values were subjected to pharmacokinetic analysis using noncompartmental methods.

The limited data obtained for the 3.0- and 10-mg/kg/day dose cohorts precluded accurate analysis of pharmacokinetic parameters beyond Cmax and Tmax, with the exception of males at 10 mg/kg/day on Day 28. For the 30-mg/kg/day dose, statistically significant differences in AUC, Cmax, and CL/F were noted between males and females on Day 28. No statistically significant differences in AUC, Cmax, Tmax, or CL/F were observed for males between Days 1 and 28, but there were statistically significant differences in AUC and CL/F for females between Days 1 and 28.

For males, following Day 1 TDF administration, TFV plasma concentrations reached mean peak values of 0.864, 0.487, and 3.45 g/mL at the 3.0-, 10-, and 30-mg/kg/day dose levels, respectively. Across the dose groups, the mean Tmax values in plasma ranged from 0.313 to 0.750 hour. For the 30-mg/kg/day dose group, TFV plasma concentrations declined in a biphasic manner with an apparent terminal half-life of 11.8 hours. The mean AUC values remained similar, from 11.2 5.35 g·h/mL (AUCinf) on Day 1 to a steady state level of 14.4 2.35 g·h/mL (AUC0 t) on Day 28. In addition, Cmax values remained similar following repeat dosing within each dose group. These data suggest linear pharmacokinetics upon repeat dosing.



In comparison, for females, following Day 1 TDF administration, TFV plasma concentrations reached mean peak values of 0.459, 0.985, and, 2.39 g/mL at the 3.0-, 10-, and 30-mg/kg/day dose levels, respectively. Across the dose groups, mean Tmax values in plasma ranged from 0.313 to 0.750 hour and Cmax increased in a dose-proportional manner on Day 1. For the 30-mg/kg/day dose group, TFV plasma concentrations declined in a biphasic manner with an apparent terminal half-life of 10.7 hours. The mean AUC values increased from 7.95 1.94 g·h/mL (AUCinf) on Day 1 to a steady state level of 24.6 6.12 g·h/mL (AUC0 t) on Day 28, indicating nonlinear pharmacokinetics upon repeat dosing. For females, TFV Cmax increased in a greater than dose-proportional manner on Day 28, indicating nonlinear pharmacokinetics between the dose groups at steady state.

2.6.6.3.4.7. Tenofovir DF: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 (bis-POC PMPA; PMPA Prodrug) in the Beagle Dog (98-TOX-4331-003)

The toxicity of TDF (GS-4331-05; bis-POC PMPA: PMPA pro-drug) in male beagle dogs (4 per group) was evaluated following oral gavage administration during daily, BID, every other day, or weekly oral administration to the dog over a 28-day treatment period at dosages of 0, 30, 15, 60, or 210 mg/kg/dose, respectively (Tabulated Summary 2.6.7.7.R,98-TOX-4331-003). The dogs in the vehicle control group received 50 mM citric acid BID for 28 consecutive days. Two of the 4 dogs in the 210 mg/kg once-weekly group became moribund and were euthanized on Day 5 or 6 after receiving only 1 dose on Day 1. Transient body weight losses and decreased body weight gains occurred in all dose groups and reduced food consumption in the 60 and 210 mg/kg groups. There were marginal to slight increases in creatinine, total bilirubin, and triglycerides (except at 210 mg/kg) and increased urine volume in all dose groups. For the 2 dogs euthanized moribund on Day 5 or 6, there were numerous changes in clinical chemistry parameters. At necropsy, pale kidneys and decreased absolute/relative thymus weights were noted in all dose groups. Slight to moderate renal tubular karyomegaly, degeneration/regeneration, single cell necrosis, and mononuclear cell infiltration were observed in dogs in all dose groups surviving to Day 29. Moderate to severe renal tubular cell necrosis (diffuse) and mineralization were noted in the 2 dogs euthanized moribund on Day 5 or 6. In summary, no obvious differences in this study were observed in the effects produced by TDF in beagle dogs on a daily, BID, or twice weekly dose schedule (total dose of 840 mg/kg/group) during a 28-day treatment period. Greater effects were observed when TDF was administered as a single dose of 210 mg/kg.


Blood samples were obtained prior to TDF dosing and at 0.5, 1, 2, 4, 6, 12, and 24 hours postdose on Day 1 and during Week 4 (Tabulated Summary 2.6.5.4.HH, 98-TOX-4331-003-PK). Blood samples (predose and 1 hour) were also taken on Days 7/8, 15, and 21/22. Plasma samples were analyzed using a validated reverse-phase, ion-pair HPLC method with fluorescence derivatization.

Following the first day of TDF administration, concentrations of TFV in plasma reached mean peak values of 2.64, 4.48, and 8.04 g/mL at dose levels of 15 (BID), 30 (once daily), and 60 (every other day) mg/kg, respectively. For the group receiving 210 mg/kg (once



weekly), 2 dogs developed renal failure and were euthanized on Days 5 and 6 prior to receiving a second dose. These dogs showed higher overall Day 1 TFV plasma concentrations, with a mean Cmax value of 49.8 g/mL relative to 26.9 g/mL for the remaining dogs in the once-weekly dose schedule. Tenofovir plasma concentrations reached Cmax at 0.5 to 1.0 hour and declined in a biphasic manner. Following the last day of oral administration of TDF, TFV plasma concentrations reached maximum values within 0.75 to 1.4 hours postdose, with mean peak values of 7.54, 13.0, and 32.9 g/mL for the dose schedules of 30 (once daily), 60 (every other day), and 210 (once weekly) mg/kg , respectively. Pharmacokinetic parameters for the BID group could not be evaluated due to inadequate sampling.

Comparison of dose normalized Cmax with dose suggested a decrease in Cmax with increasing dose that appears to correspond with a delay in Tmax. Dose schedules once daily, every other day, and once weekly yielded mean terminal half-life values of 29.4, 41.6 and 40.1 hours, with AUCtau values of 14.9, 32.2, and 134 g·h/mL, respectively. Comparison of dose normalized AUCtau values with dose demonstrated dose proportionality, indicating dose linear TFV exposure across dose schedules at steady state. However, this interpretation may be incorrect at the higher dose level due to premature termination of 2 of 4 dogs on the once weekly schedule and only reflects linear pharmacokinetics in the surviving animals in this group. Comparison of AUCinf on Day 1 with AUCtau at steady state across dose schedules shows a general trend of approximately 2-fold greater exposure at steady state than following the first dose. These data suggest that repeat dosing in dogs at this dose level results in changes in TFV plasma clearance with time.

2.6.6.3.4.8. Tenofovir DF: A 13 and 42-Week Oral Gavage Toxicity Study (with a 13-Week Recovery Period) of bis poc-PMPA (GS-4331-05) in the Beagle Dog (97-TOX-4331-001)

The toxicity of TDF (bis-POC PMPA; GS-4331-05) in beagle dogs was evaluated following oral gavage administration once daily for 13 or 42 consecutive weeks at doses of 0 (vehicle), 3, 10, or 30 mg/kg/day (Groups 1–4, respectively), followed by a 13-week drug free recovery period (Tabulated Summary 2.6.7.7.S, 97-TOX-4331-001). Blood samples were taken on study Day 1 and Weeks 13, 26, and 42 to assess systemic drug exposure. Toxicity associated with test article administration was evaluated by monitoring clinical signs of toxicity (BID), detailed physical examinations (weekly), body weights (weekly), food consumption (daily), ophthalmology (Weeks 13, 26, 42 and 55), and electrocardiographic evaluations (Weeks 6, 13, 26, 42, and 55). In addition, evaluation of clinical biochemistry, hematology, and urinalysis values (Weeks 6, 13, 26, 42, 48, and 55/54); and markers of bone turnover (added after initiation of the study, including urine [Weeks 8/7, 12, 25, 41, 47, and 54] and serum [Weeks 8/7, 13, 26, 42, 48, and 55/54]) were performed. Gross necropsy findings, organ weight changes, histopathology, and pQCT (bone densitometry) (Weeks 14, 42, and 55) were performed.

There was 1 death (control male) during the treatment period, which was likely caused by a gavage accident that resulted in fibrinohemorrhagic bronchopneumonia. Body weight loss was observed in animals treated in the 10 (males only during the first week of dosing) and



30 mg/kg/day dose groups during the first 2 weeks of dosing and, although subsequent weight gains appeared to be similar to the vehicle control group, mean values for these groups remained marginally lower throughout the remainder of the dosing and recovery periods. There were no treatment-related clinical signs of toxicity or effects on food consumption, and no treatment-related ophthalmologic or electrocardiographic findings. Although there were statistically significant increases in APTT values in the 30-mg/kg/day dose group during the dose period, the APTT values were within normal biological limits. No other treatment-related hematological changes were observed in this study. Treatment-related changes in clinical biochemistry parameters included slightly increased total bilirubin and chloride concentrations, ALP, and creatine kinase activities; and slight to moderate decreases in cholesterol, bicarbonate, and potassium concentrations. There was a partial return to normal or evidence of recovery for potassium, chloride, total bilirubin, and ALP values after the recovery period (Week 55/54). Although within normal biological limits, the creatinine was minimally increased and the glucose was minimally decreased (Week 13). Glucosuria, proteinuria, and increased urinary output were observed in the 30-mg/kg/day dose group throughout the study. Glucosuria was still evident in the urine of 2 of 4 animals at Week 55/54.

Serum N-telopeptide, a bone resorption marker, was generally increased at all time points evaluated during the treatment period for dogs in the 30-mg/kg/day dose group and remained increased at the end of the recovery period (Week 54). Increased urinary calcium and phosphorus levels; increased bone specific ALP levels; and decreased 1,25 vitamin D3 levels were also observed in the 30-mg/kg/day dose group during the treatment period. Serum N-telopeptide, bone specific ALP, and 1,25 vitamin D3 levels did not approximate control values by the end of the recovery period.

Absolute and relative kidney weights in dogs in the 30-mg/kg/day dose group were generally higher than controls (without necessarily attaining statistical significance) at both necropsy examinations (Weeks 14 and 42). Gross examination revealed pale discoloration of the renal cortex in several animals in the 10- and 30-mg/kg/day dose groups at the Weeks 14, 42, and 55 (recovery) sacrifices. There was a trend for low liver weights in males (30 mg/kg/day) at Weeks 14, 42, and 55. Dose-related histopathologic changes included slight to mild renal tubular dilatation or degeneration/regeneration and interstitial nephritis in the 10- and/or 30-mg/kg/day dose groups at Weeks 14, 42, and 55. Slight to moderate renal tubular karyomegaly was observed in the 3-, 10-, and 30-mg/kg/day dose groups at Weeks 14, 42, and 55 (except 3 mg/kg/day, not observed at end of the 13-week recovery period). In the liver, slight centrilobular sinusoidal dilatation/congestion and/or cell pigment accumulation were noted in the 30-mg/kg/day dose group at Weeks 14, 42, and 55.



The pQCT evaluation indicated that there was a marginal/slight decrease in bone mineral content and bone mineral density of the total slice and trabecular and cortical areas of the distal femur metaphysis of males and females (30 mg/kg/day) at 13 and 42 weeks, with some evidence of recovery at Week 55. Decreases in cortical thickness generally correlated with slight decreases in periosteal circumference and slight increases in endosteal circumference. Minimal effects on the cortex were observed at the mid-femur diaphysis. Statistically significant decreases in cortical thickness and increases in endosteal circumference were observed at 30 mg/kg/day (females only) at Week 42. Cortical area and bone mineral content were statistically significantly decreased at Week 42 at 30 mg/kg/day (males only). There were no adverse effects at 3 or 10 mg/kg/day on bone mineral content or bone mineral density. Changes in tested biochemical markers of bone turnover (increased urinary N-telopeptide; increased urinary calcium and phosphorus; increased bone specific ALP; and decreased 1,25 vitamin D3) were consistent with increased bone resorption and resultant effects on pQCT-derived parameters.

In view of the renal pathology evident in all dose groups of this study, the NOEL for TDF administered daily by oral gavage to beagle dogs for 42 weeks is less than 3 mg/kg/day.


Concentrations of TFV were determined in plasma samples obtained during the 42-week repeated dose toxicity study after oral gavage of TDF in beagle dogs (Tabulated Summary 2.6.5.4.II, 97-TOX-4331-001-PK). Blood samples were obtained prior to TDF dosing, at 30 minutes and 1, 2, 4, 8, 12, and 24 hours postdose on Day 1, and at Week 13 for the Interim Sacrifice Cohort and Weeks 26 and 42 for the Main Study Cohort. A subset of these samples (from 4 males at the 3- and 10-mg/kg/day dose and 4/sex at the 30-mg/kg/day dose) was selected for analysis on Day 1 and Weeks 13 and 26. At Week 42, all 6 animals/sex/group were sampled, including 2 animals/sex/group designated for recovery. Plasma samples were assayed for TFV using a validated reversed-phase ion-pair HPLC method with fluorescence derivatization. Plasma TFV concentration values were subjected to pharmacokinetic analysis using noncompartmental methods. Due to insufficient plasma concentration data over time, median pharmacokinetic parameters were limited to Cmax and Tmax for Day 1 and Weeks 13 and 26 for the 3 mg/kg/day dose group and Day 1 for the 10 mg/kg/day dose group. No statistical differences in AUC, Cmax, or Tmax were observed between genders. Following TDF administration on Day 1, concentrations of TFV in plasma reached median peak values of 0.254, 0.665, and 2.46 g/mL at the 3-, 10-, and 30-mg/kg/day dose levels, respectively. The Tmax in plasma ranged from 0.5 to 1 hour and plasma concentrations declined in a biphasic manner, with an apparent half-life of 18.8 hours (30 mg/kg/day cohort). Tenofovir Cmax appeared to increase in a dose linear manner following Day 1 TDF administration. Following repeat dosing, AUC values increased from 11.5 g·h/mL (AUCinf) on Day 1 to steady state levels of 29.0 to 29.8 g·h/mL (AUC0 t) at Weeks 13 and 42 for the 30-mg/kg/day TDF dose cohort. These data suggest nonlinear pharmacokinetics upon repeat dosing. Alternatively, this increase may be due to an underestimate of Day 1 AUC due to inadequate assessment of the terminal half-life and corresponding pharmacokinetic parameters. Also, TFV Cmax and AUC0 t increased in a



greater than dose proportional manner at Weeks 13, 26, and 42, indicating nonlinear pharmacokinetics between dose groups at steady state.

2.6.6.3.4.9. Emtricitabine/Tenofovir DF: 4-Week Oral Gavage Toxicity and Toxicokinetic Study with Emtricitabine/Tenofovir Disoproxil Fumarate (FTC/TDF) in Male Dogs with a 4-Week Recovery Period (TX-164-2004)

The purpose of this study was to evaluate the toxicity and to determine the toxicokinetics of the test articles FTC and TDF, alone or in combination, when administered daily via oral gavage to dogs for at least 4 weeks and to assess the reversibility, persistence, or delayed occurrence of any effects after a 4-week recovery (Tabulated Summary 2.6.7.7.T, TX-164-2004).

Male purebred beagles were assigned to 5 groups (n = 4/group) and administered vehicle (50 mM citrate buffer in reverse osmosis water), 20 mg/kg/day FTC, 30 mg/kg/day TDF, 2/3 mg/kg/day FTC/TDF, or 20/30 mg/kg/day FTC/TDF in combination. Two additional animals administered 20/30 mg/kg/day FTC/TDF were designated for recovery sacrifice and underwent 4 weeks of recovery following 29 days of treatment.

Assessment of toxicity was based on mortality, clinical observations, ophthalmic examinations, body weights, food consumption, and clinical and anatomic pathology. Samples were also collected for toxicokinetic evaluation. In addition, immunophenotyping and natural killer (NK) cell analysis were conducted.

All animals survived to scheduled sacrifice. No clear test article-related effects on clinical signs and ophthalmic examinations were observed. Decreases in body weight gain and food consumption were noted in animals given 30 mg/kg/day of TDF and 20/30 mg/kg/day of FTC/TDF respectively; however, these were not statistically significant.

Between Days 1 and 27, no accumulation of FTC occurred when FTC was administered alone at 20 mg/kg/day or in combination with 30 mg/kg/day TDF. The mean AUC values for Days 1 and 27 were 20.85 and 25.28 g·h/mL, respectively, when administered alone and 24.46 and 32.33 g·h/mL, respectively, when administered in combination. During the same period, no unexpected accumulation of TFV occurred when TDF was administered alone at 30 mg/kg/day or in combination with 20 mg/kg/day FTC. The mean AUC values for Days 1 and 27 were 7.60 and 18.41 g·h/mL, respectively, when administered alone and 8.85 and 20.87 g·h/mL, respectively, when administered in combination.

TDF administration at 30 mg/kg/day, alone or in combination with FTC administration, was associated with minimally increased APPT and creatinine. Both effects appeared to be reversible following recovery. Urinalysis test results appeared unaffected.

No remarkable changes were observed for immunophenotyping or NK cell assay values for test article-treated groups as compared with the vehicle control group.



No test article-related macroscopic findings or organ weight variations were present at either the terminal or recovery sacrifice.

Test article-related microscopic findings at the terminal sacrifice were seen in the kidneys of animals in 2 groups. Minimal tubular epithelial necrosis and an increased incidence and mean severity of tubular epithelial regeneration were seen in animals given 30 mg/kg/day of TDF alone or in combination with 20 mg/kg/day of FTC. Kidney lesions were morphologically similar in the 2 affected groups, and there were no overall differences in incidences and mean severities of tubular necrosis and regeneration noted between the 2 groups.

No test article-related microscopic changes were observed at the recovery sacrifice, indicating that the kidney findings noted microscopically at dosing termination were reversible.

In conclusion, administration of FTC alone at a dose of 20 mg/kg/day, or FTC/ TDF combination at 2/3 mg/kg/day, was well tolerated by dogs for 4 weeks. TDF alone at 30 mg/kg/day and FTC/TDF at 20/30 mg/kg/day resulted in microscopic kidney lesions, which in the case of FTC/TDF at 20/30 mg/kg/day were reversible after a 4-week recovery period. Based on the clinical and anatomic pathology findings, the NOAEL was 20 mg/kg/day for FTC alone and 2/3 mg/kg/day for the FTC/TDF combination. A NOAEL for TDF could not be determined at the doses used in the study.

2.6.6.3.5. Monkey

2.6.6.3.5.1. Emtricitabine: A 30-Day Oral Toxicity Study with TP-006 in Cynomolgus Monkeys Given 524W91 (TOX600)

In a 1-month oral toxicity study, cynomolgus monkeys (5/sex/dose) were given FTC at 0, 80, 400, or 2000 mg/kg/day in 0.5% methylcellulose, as equal divided doses 6 hours apart via nasogastric intubation for 28 days, after which 3/sex/dose were necropsied (Tabulated Summary 2.6.7.7.U, TOX600). Remaining monkeys (2/sex/dose) were held without dosing for 3 weeks, then necropsied to evaluate recovery from treatment-related effects. The following parameters were monitored for signs of toxicity: clinical signs, appetite, body weights, ophthalmology, electrocardiography, neurophysiology, hematology, clinical chemistry, urinalysis, plasma drug concentrations, cerebrospinal fluid (CSF) drug concentrations, organ weights, gross pathology and histopathology, and neurohistopathology. Samples of skeletal and cardiac muscle from control and high dose animals were examined by transmission electron microscopy.



There were no changes in any of the observed parameters suggestive of treatment-related effects. The only possibly treatment-related clinical sign was an increased incidence of soft feces in females at 2000 mg/kg/day. Systemic exposure to FTC was similar between sexes and between Days 3 and 27. The Cmax and the AUC values increased in proportion to the dose. No significant drug accumulation occurred over the dosing period. Drug levels in CSF measured 1 hour after dosing in the third week of the study (together with a companion blood sample) demonstrated that CSF drug levels rose proportionately with plasma drug levels and were 4.3%, 3.7%, and 3.7% of plasma drug levels in the 80-, 400-, and 2000-mg/kg/day groups, respectively. A NOEL of 400 mg/kg/day was established; corresponding systemic exposure levels (AUC0 6h) at 3 days were 687 to 688 M·h (148 g·h/mL) for males and females, respectively, and at Day 27 were 793 to 664 M·h (170 to 143 g·h/mL) for males and females, respectively.

2.6.6.3.5.2. Emtricitabine: A 3-Month Oral Toxicity Study in Cynomolgus Monkeys Given 524W91 (TOX627)

In a 3-month GLP oral toxicity study, cynomolgus monkeys (5/sex/dose) were given FTC at 0, 40, 200, or 1000 mg/kg/day in 0.5% methylcellulose, as equal divided doses 6 hours apart for 3 months, then 3/sex/dose were killed (Tabulated Summary 2.6.7.7.V, TOX627). The tissues were fixed by perfusion and the monkeys were necropsied. Remaining monkeys (2/sex/dose) were allowed to recover for 3 weeks, and evaluated for reversibility of treatment-related effects. The following parameters were observed for signs of toxicity: clinical signs, body weight, ophthalmology, electrocardiography, neurophysiology and neurohistopathology, hematology, clinical chemistry, urinalysis, and plasma drug concentrations. At terminal necropsy, organs were weighed and gross and histopathology were performed. Liver, cardiac, and skeletal muscle was examined by transmission electron microscopy. There were no treatment-related changes in any of the observed parameters at any dose. A NOEL of 1000 mg/kg/day was established. Cmax and AUC increased with dose and there was no evidence of accumulation. Systemic exposure to FTC was similar between sexes and between Days 3 and 87. In the 1000-mg/kg group, AUC0 6h values on Day 3 for males and females were 1339 and 942 M·h (288 and 203 g·h/mL), respectively, and on Day 87 were 1630 and 1109 M·h (350 and 238 g·h/mL), respectively.



2.6.6.3.5.3. Emtricitabine: 52-Week Oral Toxicity Study of FTC (Emtricitabine) in Cynomolgus Monkeys with a 4-Week Recovery Period (TOX032)

In a 1-year oral toxicity study with a 3-month interim necropsy, cynomolgus monkeys were given FTC at 0, 50, 200, or 500 mg/kg/day in 0.5% methylcellulose (n = 8, 4, 4, and 8 males and females, respectively) as equal divided doses at least 5 hours apart, by nasogastric intubation, daily for 52 weeks (Tabulated Summary 2.6.7.7.W, TOX032). After dosing for 3 months, 2 monkeys/sex at 0 and 500 mg/kg/day were necropsied. At the end of dosing, 4 monkeys/sex/dose were necropsied. Remaining monkeys (2/sex at 0 and 500 mg/kg/day) were allowed to recover for a month and evaluated for reversibility of treatment-related effects. Data collected were body weights, food consumption, clinical signs, ophthalmic examinations, ECG recordings (with measurement of QT interval), neurophysiology parameters, hematology, clinical chemistry, plasma FTC concentrations (Day 1 and Weeks 13, 26, and 52), gross pathology, organ weights, and histopathology findings.

There were no signs of toxicity after dosing for 3 months at any dose. At the end of 52 weeks of dosing, the only treatment-related effects were a slight (nonsignificant) decrease in RBC count and increased MCH at 500 mg/kg/day in females only, which were absent at the end of the recovery period. No other signs of toxicity occurred at any dose. Based on these findings, the NOEL was 200 mg/kg/day.

Emtricitabine was rapidly and well absorbed with peak plasma concentrations occurring between 0.5 to 2 hours postdosing. Plasma half-life was 2 to 4 hours for all dose levels. Half-life may have been underestimated because of the short (6 hour) sampling period. A longer half-life was expected, based on the measurable predose drug concentrations and the slightly higher plasma FTC concentrations after repeated dosing. No major differences were noted in plasma FTC exposure between male and female monkeys. Plasma Cmax, AUC0 6h, and estimated steady-state AUC0 24h (Weeks 13, 26, and 52) increased linearly over the dose range of 50 to 500 mg/kg/day. Systemic exposure to FTC was similar between sexes and at Weeks 13, 26, and 52. Week 52 AUC0 24h values at the NOEL of 200 mg/kg/day were 97.2 and 97.8 g·h/mL in males and females, respectively. Corresponding Cmax values were 17.4 and 21.3 g/mL, respectively.

2.6.6.3.5.4. Rilpivirine: 8-Week Oral Toxicity Study in Juvenile Female Cynomolgus with TMC278.HCl

Nonhuman primates, like man, have a significant adrenal androgenic pathway which is not present in rodents and dogs. For that reason, the effects of TMC278 on that pathway were evaluated in cynomolgus monkeys. Estrous cycle physiology in cynomolgus monkeys resembles that of man better than dogs. Consequently, this nonhuman primate species at an age prior to puberty was also used to evaluate the early ovarian maturation observed in immature dogs.



The doses for the toxicity study in cynomolgus monkeys were selected on the basis of a toxicokinetic study described in the Pharmacokinetics Written Summary, Module 2.6.4, Section 2.6.4.3.4.8. For the repeat dose part of that study (TMC278-NC326), the animals were habituated to restraining chairs for ECG recordings. TMC278 was administered for 15 days to 3 groups of 3 young mature cynomolgus monkeys at 0 (vehicle), 200, or 500 mg/kg/day. Recordings were made on Days 2 and 15. No abnormalities were noted on RR, PR, or QT (also corrected according to Fridericia {15639}) intervals, on the form of the QRS complex, or the T-wave. Moreover, no treatment-related effects were noted on the systolic, diastolic, or mean arterial blood pressure. Mean heart rate at all times was similar in all groups, ranging from 226 to 248 beats/minute.

TMC278 suspended in 1% m/v aqueous HPMC with 0.5% Tween 20 was administered for 8 weeks to 3 groups of 8 immature female cynomolgus monkeys at 0 (vehicle), 200, or 500 mg/kg/day. Animals were dosed 0, 100, or 250 mg/kg BID, with a 6-hour interval, at a volume of 5 mL/kg (Tabulated Summary 2.6.7.7.X, TMC278-NC248). Regular observations of clinical signs, menses, morbidity, and mortality were made. Weekly, body weights were determined. Blood samples were taken for clinical pathology including hematology, clinical chemistry, and urinalysis prior to the start of dosing and towards the end of the dosing period. Blood samples for endocrinology (progesterone, estradiol, and LH) were taken prior to the start of the dosing period and weekly prior to the TMC278 administration (at approximately the same time as the samples were taken during the dosing period). Adrenocorticotropic hormone stimulation tests including determination of serum levels of ACTH (only prior to challenge), cortisol, 17 -hydroxyprogesterone, progesterone, androstenedione, and dehydroepiandrosterone (DHEA) prior to ACTH challenge and 15, 30, and 60 minutes after challenge were done prior to the start of the dosing period and 2, 4, and 6 weeks after the start of dosing. Samples for toxicokinetics were taken the day before necropsy at 10 time points over a 24-hour period. At necropsy at the end of the dosing period, gross lesions were recorded. A range of organs was weighed and a complete set of organs and tissues was examined microscopically.

No mortalities occurred in this study. No adverse or relevant effects were observed on body weight, clinical pathology, organ weights, or gross lesions. In (trough level) samples taken prior to the daily dosing, increased levels of 17 -hydroxyprogesterone (up to 100%) and progesterone (up to 57%) were noted throughout the dosing period in a more or less dose-related fashion. Reduced concentrations of androstenedione (maximally 31%) were evident throughout the dosing period in both dose groups to a similar extent. Decreased DHEA concentrations (maximally 20%) were noted only in the group dosed with 500 mg/kg/day after 4 and 6 weeks of dosing. No effects were noted on cortisol and ACTH in these samples taken at TMC278-trough level.

Upon ACTH challenge, serum concentrations of 17 -hydroxyprogesterone showed a very strong dose-related response throughout the dosing period, with Cmax values of the groups treated with 200 and 500 mg/kg/day being 2.5- and 4-fold higher, respectively, than in the control group at 4 and 6 weeks of treatment. A similar pattern was visible for progesterone upon challenge. Cmax values of the groups treated with 200 and 500 mg/kg/day were 2.5- and 3-fold higher, respectively, than in the vehicle group. Androstenedione and DHEA levels in



the vehicle group showed only limited response to challenge. The Cmax values of androstenedione and DHEA at 500 mg/kg/day were maximally 50% lower. Cortisol Cmax values at 500 mg/kg/day were maximally 56% lower at the end of the dosing period.

Only incidental vaginal bleedings not indicative of menses were recorded. Moreover, no follicular or ovulatory effects were noted on serum levels of progesterone, estradiol, or LH. Microscopic evaluation of ovaries did not show any indication of activation. Minimal follicular cell hypertrophy in the thyroid gland was scored in 1 control animal, 3 animals dosed with 200 mg/kg/day, and 4 animals dosed with 500 mg/kg/day.

Since at the lowest dose tested several hormonal effects were evident, no NOAEL was established. Systemic exposure expressed as Cmax and AUC values are given in Table 17 below (Tabulated Summary 2.6.7.7.X, TMC278-NC248).

Table 17. Exposure to TMC278 (Cmax and AUC) in an 8-Week Oral Study with TMC278 in Juvenile Female Cynomolgus Monkeys

Dose (mg/kg/day) Sampling Day Cmax1 (μg/mL) Cmax2 (μg/mL) AUC0 24h (μg.h/mL)

200 Day 55 0.14 0.18 2.7

500 Day 55 0.31 0.31 4.6

Cmax1 = Cmax after first daily dose, Cmax2 = Cmax after second daily dose

2.6.6.3.5.5. Tenofovir DF: A 56-Day Study of Tenofovir DF Administered Orally and of PMPA Administered by Subcutaneous Injection to Rhesus Monkeys (P2000078)

Findings from this mechanistic study are summarized in Section 2.6.6.8.3.3.6.


Administration of FTC/TDF in combination is unlikely to exacerbate known toxicities of the individual agents. This was confirmed by the absence of any new or more marked toxicities in a 14 day rat toxicology study of the FTC/TDF tablet in comparison with studies of a similar duration with the individual agents and a 4-week dog study with the combination (Tabulated Summaries 2.6.7.7M and 2.6.7.7.T, TX-164-2001 and TX-164-2004, respectively). In addition, coadministration of TDF and FTC to SIV-infected pig-tailed monkeys at dose levels many times higher than the intended clinical dosages for up to 6 months had no obviously deleterious effects on the animals (Tabulated Summary 2.6.3.1.4, {5477}).



The repeat dose toxicity studies with RPV (TMC278) have identified a series of targets of toxicity: RBC in mouse, rat, and dog; coagulation in rat; liver in rat and dog; kidneys in mouse and dog; thyroid gland with secondary effects on the pituitary gland in rat; adrenal glands in mouse, rat, dog, and cynomolgus monkey; testes in dog; ovaries in dog; and in immature females, secondary effects on other parts of the genital tract and on mammary glands compared to immature control animals. The majority of these effects appeared to be completely reversible after a recovery period. The effects on thyroid gland and coagulation in rats showed signs of recovery but this was not complete at the end of the 1-month postdosing period. For mice and rats, a NOAEL could be established. However, dogs and cynomolgus monkeys showed effects at the level of the adrenal glands at the low dose tested. Moreover, dogs also showed effects on ovaries in almost all pivotal studies at the low dose given. These effects prevented establishment of NOAELs in these species.

The 3 drugs, FTC, RPV and TDF, exhibit different patterns of target organ toxicity. Specifically, the only significant effect of FTC identified at dose levels constituting large clinical multiples was a minor anemia. In contrast, extensive nonclinical investigations of the toxicity of TDF have shown that the bone marrow is not a target for this agent, and that the target organs for TDF are distinctly different (GI, bone, and kidney). Given that kidney effects have been observed with RPV in mice and dogs only at high dose levels and exposures, and that the routes of excretion differ for RPV and TDF, renal toxicity is not anticipated to be an issue with the FTC/RPV/TDF combination product. From in vitro data, pharmacokinetic studies in dogs (Module 2.6.4), and clinical experience (Module 2.7.2), there are no anticipated pharmacokinetic interactions between FTC, RPV, and TDF. The ample nonclinical safety database on these drugs strongly indicates that little potential for drug interaction exists and further toxicological investigations are unlikely to yield new data relevant to humans.

Further studies of longer duration with the combinations seem unwarranted given the lack of pharmacokinetic interactions and significant overlapping toxicities, and the use of additional animals that would be required to obtain such information.



2.6.6.4. Genotoxicity

2.6.6.4.1. In Vitro Studies

2.6.6.4.1.1. Emtricitabine: Mutagenicity Test with FTC in the Salmonella – Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay (18637-0-409R)

FTC was dissolved in dimethylsulfoxide (DMSO) and tested for mutagenic activity in the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay (Tabulated Summary 2.6.7.8.A, 18637-0-409R). The concentrations tested, along with vehicle and positive control test substances, were 5000, 3300, 1000, 333, and 100 g/plate in the presence and in the absence of metabolic activation. The tester strains were Salmonellatyphimurium TA98, TA100, TA1535, TA1537, and Escherichia coli strain WP2uvrA. Emtricitabine did not cause a positive increase in the number of revertants per plate in any of the tester strains in either the presence or absence of metabolic activation. The confirmatory assay, at the concentrations of FTC listed above, also gave negative results for mutagenic activity. It was concluded that FTC was negative in the reverse mutation (Ames) assay.

2.6.6.4.1.2. Emtricitabine: Salmonella/ Mammalian Microsome Assays with 524W91 (MUT203)

Emtricitabine was tested for its ability to induce reverse mutations in 2 Ames Salmonella/mammalian-microsome assays, the Salmonella plate incorporation assay and the Salmonella preincubation assay (Tabulated Summary 2.6.7.8.B, MUT203). The tester strains used were TA1535, TA1537, TA1538, TA98, and TA100, both in the presence and absence of Arochlor-induced rat liver S9 metabolic action. The range of concentrations of FTC were 100 to 5000 g/plate.

There was no detectable mutagenic activity at any dose of FTC when tested by either Ames assay method.

2.6.6.4.1.3. Emtricitabine: Mutagenicity Test of Emtricitabine using Microorganisms (K01-3154)

The ability of FTC to induce mutations was investigated by using Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537, and Escherichia coli strain WP2 uvrA with a preincubation method in the presence and absence of a metabolic activation system (S9 mix) (Tabulated Summary 2.6.7.8.C, K01-3154). The mutagenicity of the test substance was judged negative because the number of revertant colonies in the test substance groups was less than twice that of each negative control in all test strains. It is concluded that FTC has no ability to induce mutations under the conditions of the assay.



2.6.6.4.1.4. Emtricitabine: In Vitro Mammalian Cell Gene Mutation Test (TOX012)

The free base of FTC was dissolved in sterile distilled water and tested for its potential to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line (Tabulated Summary 2.6.7.8.D, TOX012). Exposures to FTC were in the presence and in the absence of Aroclor-induced rat liver S9 metabolic activation. A preliminary experiment was used to select FTC concentrations ranging from 0.5 to 5000 g/mL for testing. Cells at test concentrations of 1000, 2000, 3000, 4000, and 5000 g/mL were cloned and evaluated with and without S9. For FTC, no cloned cultures exhibited mutant frequencies which were at least 55 mutants/106 clonable cells over that of sterile distilled water. Consistent with the low order of cytotoxicity established for FTC, no toxicity in the cloned cultures, i.e., total growth of 50% of the distilled water vehicle, was observed at any dose level. The colony sizing for the methyl methanesulfonate positive control yielded the expected increase in small colonies, verifying the adequacy of the methods used to detect small colony mutants. It was concluded that FTC was negative in the L5178Y/TK mouse lymphoma mutagenesis assay at concentrations up to, and including, 5000 g/mL.

2.6.6.4.1.5. Rilpivirine: Ames Tests with Rat Liver S9

TMC278 base and TMC278 dissolved in DMSO (final concentration 0.1%) were tested in bacterial reverse gene mutation tests (Ames Test) (Tabulated Summaries 2.6.7.8.E, 2.6.7.8.F and 2.6.7.8.G, TMC278-Exp5540, TMC278-Exp5693, and TMC278-NC335, respectively). The Ames tests were done, in triplicate, with 5 histidine-requiring strains of Salmonellatyphimurium, TA98, TA100, TA1535, TA1537, and TA102. On the basis of precipitation that limited scoring in range finding studies with plate incorporation and concentrations up to 5000 μg/plate, TMC278 base was tested in 2 main studies. Each of the studies comprised 2 independent repeat assays using the plate incorporation method up to 500 μg/plate. TMC278 was studied in 1 experiment with 2 independent repeat assays, one using the plate incorporation method and the other the preincubation method, up to 250 μg/plate.

In all of these studies, TMC278 base or TMC278 tested at the highest scorable concentration did not induce a biologically significant increase (more than 2 times the frequency of the vehicle) in the mutation frequency in any of the tested strains, irrespective of the presence of metabolic activation. (Note: In the second assay of the study with TMC278 (Tabulated Summary 2.6.7.8.H, TMC278-NC279), the preincubation method was used. Under these conditions, no precipitation occurred at the highest tested concentration in the absence of S9.) In all of these studies, the positive controls gave the anticipated results, indicating the validity of the test system.

2.6.6.4.1.6. Rilpivirine: Ames Test with Human Liver S9

The metabolic pathway of TMC278 generating metabolites M30 and M31 (Figure 1) encompasses potentially reactive metabolites (epoxide) due to Michael addition at the

, -unsaturated nitrile moiety {15699} (see Figure 1 and Module 2.6.4, Pharmacokinetic Written Summary, Section 2.6.4.5).



Figure 1. Metabolites M30 (Left) and M31 (Right)

HN

N

NHN

CNHO

O

HN

N

NHN

CNHO

Usually, metabolic pathways are evaluated in in vitro genotoxicity studies due to the presence of the S9 fraction of Aroclor-induced rat liver. However, rat liver S9 appeared not to yield either of these metabolites (Tabulated Summary 2.6.5.9.C, TMC278-NC102) and for that reason it was unclear whether the metabolic pathway with the potentially reactive metabolites had been adequately evaluated. In contrast, some batches of human liver S9 did yield M30 and M31. Moreover, M30 was detected in feces at 3% of the administered dose in the human metabolism study, TMC278-TiDP6-C119 (Module 2.7.2, Clinical Pharmacology Summary, Section 2.7.2.2.2.1.4). For these reasons, TMC278 base was tested in an Ames test in the presence of human liver S9 (Tabulated Summary 2.6.7.8.H, TMC278-NC279). TMC278 base dissolved in DMSO was tested in 4 independent assays up to 500 μg/plate (the highest scorable concentration due to precipitations). The concentration of M30 + M31 (incomplete separation in liquid chromatography) indicated that the human S9 metabolic enhancer was active. The validity of the test system was evaluated by the response of positive controls used in the absence of a metabolic enhancer upon plate incorporation or preincubation, separate from each of the 4 assays. In parallel, TMC278 metabolites M30 and M31, the carboxylic and hydroxyl metabolites of the , -unsaturated nitrile moiety of TMC278, respectively, were determined by means of radiolabel LC-MS. In the first parallel assay, only the presence of M30 and M31 could be established due to the low radioactive dose. In the subsequent parallel incubations, a higher specific radioactivity of [14C]TMC278 was applied allowing the quantitative determination of the sum of M30 and M31.

It is concluded that TMC278, under conditions that it is metabolized via a pathway encompassing potentially reactive intermediates such an epoxide, did not induce a biologically significant increase of the mutation frequency. The experimental conditions in the Ames test yielded M30 + M31 at 1.4% to 2.6% of the dose applied.

2.6.6.4.1.7. Rilpivirine: In Vitro Mammalian Cell System

TMC278 base and TMC278 dissolved in DMSO were tested in mouse lymphoma L5178Y cells for their potential to induce forward mutations at the TK locus (5-trifluorothymidine resistance; Tabulated Summaries 2.6.7.8.I and 2.6.7.8.J, TMC278-Exp5539 and TMC278-NC336, respectively). Prior to the main studies, a cytotoxicity range-finding experiment was performed for both compounds.

The maximum tested concentration of TMC278 base, on the basis of precipitation that limited scoring, was 500 μg/mL for a 3-hour exposure period without metabolic activation; 35 μg/mL for a 3-hour exposure period with metabolic activation; and 100 μg/mL for a 24-hour exposure period without metabolic activation (Tabulated Summary 2.6.7.8.I, TMC278-Exp5539).



TMC278 was tested in 1 study with 2 independent repeat assays up to 50 μg/mL for a 3-hour exposure period without metabolic activation; maximally 20 μg/mL for a 3-hour exposure period with metabolic activation; and up to 50 μg/mL for a 24-hour exposure period without metabolic activation (Tabulated Summary 2.6.7.8.J, TMC278-NC336).

TMC278 base and TMC278 did not induce an increase in the number of large or small colonies of L5178Y cells. Accordingly, it was concluded that these compounds did not induce an increased mutation frequency at the TK locus of L5178Y cells or large genetic damage like chromosomal aberrations.

2.6.6.4.1.8. Tenofovir DF: Mutagenicity Test with GS-4331-05 in the Salmonella-Escherichia Coli/Mammalian-Microsome Reverse Mutation Assay

The mutagenic activity of TDF was evaluated in the Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay (Tabulated Summary 2.6.7.8.K, 96-TOX-4331-005). This assay evaluated the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonellatyphimurium tester strains and at the tryptophan locus in an Escherichia coli tester strain; both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor™-induced rat liver (S9). The doses tested in the mutagenicity assay were selected based on the results of a dose range-finding study using tester strains TAl00 and WP2uvrA and 10 doses of test article ranging from 5000 to 6.67 g per plate, 1 plate per dose, both in the presence and absence of S9 mix. The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537, and Escherichia coli tester strain WP2uvrA. The assay was conducted with 5 doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using 3 plates per dose. The doses tested were 5000, 3330, 1000, 333, and 100 g per plate in both the presence and absence of S9 mix.

The results of the Salmonella – Escherichia coli/Mammalian-Microsome Reverse Mutation Assay indicate that under the conditions of this study, TDF caused a positive increase (3.2-fold above control value) in the number of revertants per plate in 1 of 3 trials (2.4-, 3.2-, and 1.2-fold) performed with tester strain TAl535 in the absence of S9 mix. No positive increases in the number of revertants per plate were observed with any of the remaining tester strain/activation condition combinations. Therefore, GS-4331-05 was negative for mutagenic activity in this study, except in Salmonella strain TA1535 without metabolic activation, where results were equivocal based on a minimal positive response in 1 of 3 trials.



2.6.6.4.1.9. Tenofovir DF: Mutagenicity Test with GS-1278 (PMPA) Lot No. , GS-1278 (PMPA) Lot No. , GS-4331-05 (PMPA

prodrug, bis-POC PMPA) Lot No. ( ) in the Salmonella/Mammalian-Microsome Reverse Mutation Assay

The mutagenic activity of TFV (Lot numbers and ) and TDF (GS-4331-05; PMPA prodrug, bis-POC PMPA; Lot numbers [ ]) was evaluated in the Salmonella/Mammalian-Microsome Reverse Mutation Assay (Tabulated Summary 2.6.7.8.L, 97-TOX-1278-003). This assay evaluated each test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of a specific Salmonella typhimurium tester strain in the presence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor™-induced rat liver (S9).

The tester strain used in the mutagenicity assay was the Salmonella typhimurium tester strain TA1535. The assay was conducted with 6 doses of test article in the presence of S9 mix along with concurrent vehicle and positive controls using 3 plates per dose. The doses tested were 5000, 3330, 1000, 667, 333, and 100 g per plate.

The results of the Salmonella/Mammalian-Microsome Reverse Mutation Assay indicate that, under the conditions of this study, TFV Lot numbers and and TDF Lot number ( ) did not cause positive increases in the number of revertants per plate with tester strain TAl535 in the presence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).

2.6.6.4.1.10. Tenofovir DF: Mutagenicity Test of Tenofovir DF using Microorganisms

The ability of TDF to induce mutations was investigated by using Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537, and Escherichia coli strain WP2 uvrA with a preincubation method in the presence and absence of a metabolic activation system (S9 mix) (Tabulated Summary 2.6.7.8.M, K01-3037). The mutagenicity of the test substance was judged negative because the number of revertant colonies in the test substance groups was less than twice that of each negative control in all test strains. It was concluded that TDF had no ability to induce mutations under the conditions of the assay.



2.6.6.4.1.11. Tenofovir DF: Mutagenicity Test on GS-4331-05 in the L5178Y TK Mouse Lymphoma Forward Mutation Assay

The objective of this in vitro assay was to evaluate the ability of TDF (GS-4331-05) to induce forward mutations at the TK locus in the mouse lymphoma L5178Y cell line (Tabulated Summary 2.6.7.8.N, 97-TOX-4331-007). The test article formed a solution in DMSO at 500 mg/mL and remained in solution in treatment medium at all dose levels. The cytotoxicity assay was initiated with treatments up to 5000 g/mL. In the preliminary dose range-finding assay, cells were exposed to the test article from 9.85 g/mL to 5000 g/mL for 4 hours in the presence and absence of rat liver S9 metabolic activation. Under nonactivation conditions, the test article was noncytotoxic to weakly cytotoxic from 9.85 g/mL to 78.5 g/ml, and excessively cytotoxic or lethal at higher dose levels. In the presence of metabolic activation, the test article was noncytotoxic to weakly cytotoxic from 9.85 g/mL to 78.5 g/mL, and excessively cytotoxic or lethal at higher dose levels. The mutation assays were initiated with treatments based on the results of the cytotoxicity assay.

In the nonactivation assay, 7 treatments from 12.5 g/mL to 125 g/mL were analyzed for mutant induction and induced a wide range of cytotoxicity. The high dose of 125 g/mL was excessively cytotoxic and was not acceptable for evaluation. Dose-related increases in the mutant frequency were observed from 50.0 g/mL to 100 g/mL that exceeded the minimum criterion for a positive response. The increases ranged from 3.1- to 16.4-fold above the background mutant frequency. The test article was considered mutagenic in the nonactivation mutation assay. In the presence of S9 metabolic activation, 8 treatments from 12.5 g/mL to 150 g/mL were analyzed for mutant induction, and induced a wide range of cytotoxicity. Treatments at 125 g/mL and 150 g/mL were excessively cytotoxic and were not acceptable for evaluation. In the remaining 6 treatments, dose-related increases in the mutant frequency that exceeded the minimum criterion for a positive response were observed from 50.0 g/mL to 100 g/ml; the increases ranged from a 5.0- to 8.6-fold above the background mutant frequency. The test article was considered mutagenic with metabolic activation.

The test article, TDF, was therefore evaluated as positive for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under the nonactivation and S9 metabolic activation conditions used in this study.

2.6.6.4.2. In Vivo Studies

2.6.6.4.2.1. Emtricitabine: Mammalian Erythrocyte Micronucleus Test

In this study, the free base of FTC was dissolved in sterile distilled water and given by gavage to groups of 5 male ICR mice (Tabulated Summary 2.6.7.9.A, TOX011). The doses administered were 0, 500, 1000, and 2000 mg/kg. Bone marrow was aspirated from the femur at 24 and 48 hours postdose. The slides of bone marrow were stained with May-Gruenwald-Giemsa stain. Light microscopy was used to count the number of micronuclei per 2000 polychromatic erythrocytes (PCEs). The data were analyzed using a 1-way analysis of variance and pairwise comparisons were made using Dunnett’s t-test. The



positive control, cyclophosphamide (60 mg/kg by gavage), produced a marked and significant increase in micronuclei. No significant increase in micronucleated PCEs was observed, at either time point or at any dose, in mice given FTC. It was concluded that FTC was negative in the mouse in vivo micronucleus assay.

2.6.6.4.2.2. Rilpivirine: Mammalian Erythrocyte Micronucleus Test

To evaluate the clastogenic and aneuploidic effect on early forms of erythrocytes in bone marrow, TMC278 base dissolved in PEG400 + CA was administered to mice in an in vivo micronucleus test (Tabulated Summary 2.6.7.9.B, TMC278-Exp5538; Tabulated Summary 2.6.7.2.B, FK4259 [Exp 5538]). Single oral (gavage) doses of 0 (vehicle control), 100, 400, and 1600 mg/kg in 20 mL/kg were administered to 10 male and 10 female CD-1 mice per group. The dose of 1600 mg/kg was the maximally feasible dose on the basis of the maximally feasible volume for a single dose study, 20 mL/kg, and a supersaturated and highly viscous solution of 800 mg/mL. Bone marrow was sampled from 5 males and 5 females per group at 24 and 48 hours after dosing. Systemic exposure in terms of C1h and AUC0 6h was determined. A positive control group of 5 male and 5 female CD-1 mice received a single oral dose of 40 mg cyclophosphamide per kg body weight, and was sampled for bone marrow 48 hours after dosing.

TMC278 base up to a single maximal feasible dose of 1600 mg/kg did not induce structural or numerical chromosome aberrations. At this dose, no bone marrow toxicity or signs of systemic toxicity were noted. Exposure data are presented in Table 18.

Table 18. Exposure to TMC278 (C1h and AUC) in a Single Oral Dose Bone Marrow Micronucleus Study with TMC278 Base in Mice

C1h (μg/mL) AUC0 6h (μg.h/mL) Dose (mg/kg)

M (n = 3 – 5) F (n = 3 – 5) M (n = 3 – 5) F (n = 3 – 5)

100 39 40 158 130

400 59 68 262 258

1600 60 58 307 287


TMC278 did not show genotoxic potential when evaluated in a standard battery of in vitro and in vivo tests at the highest feasible concentration or dose.

2.6.6.4.2.3. Tenofovir DF: Mutagenicity Test on GS-4331-05 in the In Vivo Mouse Micronucleus Assay

The objective of this study was to evaluate the test article, TDF, for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in PCE cells in Cr1:CD-l®(1CR) BR mouse bone marrow (Tabulated Summary 2.6.7.9.C, 97-TOX-4331-008).



In the dose range-finding study, the test article was suspended in suspension vehicle and dosed by oral gavage to 3 males and 3 females per dose level. The animals were dosed at 200, 500, 800, 1500, or 2000 mg/kg and observed for 2 days after dosing for toxic signs and/or mortality. Based on the results of the dose range-finding study, the maximum tolerated dose (MTD) was estimated to be 2000 mg/kg. In the micronucleus assay, the test article was suspended in suspension vehicle and dosed by oral gavage to 6 males per dose level/harvest time point. The animals were dosed at 500, 1000, and 2000 mg/kg. The first 5 surviving animals dosed with the positive control were euthanized approximately 24 hours after dosing for extraction of the bone marrow. The first 5 surviving animals per time point dosed at the 500, 1000, and 2000 mg/kg dose level and with the vehicle control were euthanized approximately 24 and 48 hours after dosing for extraction of the bone marrow. At least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 200 erythrocytes for each animal. An additional 5 animals/dose level for the 2000, 1000, and 500 mg/kg dose levels were dosed as satellite animals for blood collection. Approximately 1 hour after dosing, blood samples were collected from the animals in the satellite groups. Shortly prior to euthanasia for bone marrow collection, blood samples were collected from the first 5 surviving animals in each group, except for the positive and vehicle control animals.

The test article, TDF, induced no signs of clinical toxicity in any of the treated animals and was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio). Tenofovir was detected in the plasma at all dose levels tested. Tenofovir DF did not induce a statistically significant increase in micronuclei in bone marrow PCEs and is considered negative in the mouse bone marrow micronucleus test under the conditions of exposure in this assay.

Pharmacokinetic Evaluation (97-TOX-4331-008 PK)

Levels of TFV were determined in mouse plasma samples obtained during an in vivo micronucleus assay (Tabulated Summary 2.6.5.3.N, 97-TOX-4331-008 PK). Plasma samples (5 mice/dose/time point) were obtained 1, 24, and 48 hours following dosing. Plasma TFV levels were determined using a validated HPLC method with fluorescence detection. At 1 hour after dosing, plasma TFV concentrations were dose proportional. Mean plasma TFV concentrations at doses of 500, 1000, and 2000 mg/kg were 3.73, 8.13, and 28.18 g/mL, respectively. At 24 hours, mean plasma TFV levels remained dose proportional. Plasma TFV levels were 0.68, 1.21, and 2.06 g/mL at dose levels of 500, 1000, and 2000 mg/kg, respectively. At 48 hours, mean plasma levels were no longer dose proportional. Plasma TFV levels were 0.045, 0.152, and 0.921 g/mL at dose levels of 500, 1000, and 2000 mg/kg, respectively. The limited plasma data precluded determination of plasma half-life and AUC. However, from the plasma concentration data it was apparent that greatest exposure ( 85% of total) to TFV occurred during the initial 24-hour period. Since plasma TFV levels were dose proportional during this period, the overall exposure to TFV during the micronucleus assay was most likely dose proportional.



2.6.6.4.2.4. Tenofovir DF: In Vivo/In Vitro Unscheduled DNA Synthesis in Rat Primary Hepatocyte Cultures at 2 Time Points

The objective of this study was to detect DNA damage caused by TDF, or an active metabolite, by measuring UDS in vivo in rat primary hepatocytes cultured in vitro (Tabulated Summary 2.6.7.9.D, 23291-0-494-OECD). For the UDS assay, the test article was administered as a single oral gavage dose in volumes of 20 mL/kg to male rats at doses of 500, 1000, and 2000 mg/kg in a preparation of 0.5% high viscosity carboxymethyl cellulose/0.8% benzyl alcohol/0.5% polysorbate 80. The hepatocytes were harvested 2 to 4 hours or 14 to 16 hours after dosing. Vehicle control groups treated with 20 mL/kg of vehicle were run concurrently at each time point. Positive control groups were treated with single IP doses of 10 mg/kg or 15 mg/kg of dimethylnitrosamine (DMN) and the hepatocytes were harvested 2 to 4 hours or 14 to 16 hours after dosing, respectively. Four male Fischer rats were treated per group.

One animal treated in the 2000 mg/kg group at the 14 to 16 hour time point was found to have a red crust around the nose and mouth prior to perfusion. All other animals at all other time points were normal. Four animals from each group were perfused and continued through attachment. Three cultures per group were evaluated for UDS.

At 2.3 to 2.9 hours or 14.4 to 15.5 hours after a single dose with the test article, primary hepatocyte cultures were prepared. After attachment of the cells, the cultures were labeled with 10 Ci/mL [3H]TdR for 4 hours. The cultures were prepared for analysis of nuclear labeling after removal of the radioactivity and addition of 0.25 mM thymidine. After autoradiography, all 3 treatment groups from each time point were selected for analysis of nuclear labeling. Dose levels of 500, 1000, and 2000 mg/kg were therefore used to interpret the UDS response at both the 2 to 4 hour and 14 to 16 hour time points.

The minimum criteria for a positive UDS response for the 2 to 4 hour time point were a mean nuclear grain count 2.47 and/or 13.33% of the nuclei containing 5 net nuclear grains (NNG). None of the treatments induced a 2.47 mean nuclear grain count, but treatment at 2000 mg/kg induced an increase of nuclei containing 5 NNG to 17.78%. For the 14 to 16 hour time point, the minimum criteria for a positive UDS response were a mean nuclear grain count 2.81 and/or 15.78% of the nuclei containing 5 NNG. None of the treatments induced a 2.81 mean nuclear grain count. However, treatment at 1000 mg/kg showed 17.11% of the nuclei containing 5 NNG and the 2000 mg/kg treatment showed 23.66% of the nuclei containing 5 NNG.

Since only 1 criterion was met at the 2 time points, TDF was evaluated as weakly positive in the in vivo/in vitro assay for UDS in rat primary hepatocyte cultures at 2 time points.



2.6.6.4.3. Emtricitabine/Tenofovir DF

2.6.6.4.3.1. Emtricitabine/Tenofovir DF: Bacterial Reverse Mutation Assay with Emtricitabine/Tenofovir Disoproxil Fumarate

A combination of FTC and TDF was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9 (Tabulated Summary 2.6.7.8.O, TX-164-2002). The assay was performed in 2 phases using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article. Dimethylsulfoxide was selected as the solvent of choice based on compatibility with the target cells. Dosing solutions of the 2 test article components were prepared in a fixed ratio of 2 parts of FTC to 3 parts of TDF. Each component was dissolved separately in DMSO and then combined. Subsequent dilutions of the combination were prepared by serial dilution in DMSO. The dose levels refer to the test article combination.

The dose levels tested in the initial toxicity-mutation assay were 1.5, 5.0, 15, 50, 150, 500, 1500, and 5000 μg per plate. No positive mutagenic responses were observed. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the initial toxicity-mutation assays, the dose levels tested in the confirmatory mutagenicity assay were 50, 150, 500, 1500, and 5000 μg per plate. In the confirmatory mutagenicity assay, no positive mutagenic response was observed. Neither precipitate nor appreciable toxicity was observed. Under the conditions of this study, the combination of FTC and TDF were concluded to be negative in the Bacterial Reverse Mutation Assay.

2.6.6.4.3.2. Emtricitabine/Tenofovir DF: In Vitro Mammalian Cell Gene Mutation Test (L5178Y/TK+/- Mouse Lymphoma Assay) with Emtricitabine/Tenofovir Disoproxil Fumarate

The test article, FTC and TDF at a fixed ratio of 2:3 (FTC/TDF), was tested in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay in the absence and presence of Aroclor-induced rat liver S9 (Tabulated Summary 2.6.7.8.P, TX-164-2003). The preliminary toxicity assay was used to establish the concentration range for the mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test article. Dimethylsulfoxide was selected as the solvent of choice based on solubility information and compatibility with the target cells.



In the preliminary toxicity assay, the maximum concentration of FTC/TDF was 3575 μg/mL. No visible precipitate was present at any concentration in treatment medium; however, substantial toxicity (i.e., suspension growth of 50% of the solvent control) was observed at

500 μg/mL with and without S9 activation (4-hour exposure) and at 150 μg/mL without activation (24-hour exposure). Based on the results of the preliminary toxicity assay, the concentrations chosen for treatment in the mutagenesis assay ranged from 15 to 1000 μg/mL for the nonactivated cultures and 15 to 1500 μg/mL for the S9-activated cultures. No visible precipitate was present at any concentration in treatment medium. The concentrations chosen for cloning were 15, 50, 100, 150, and 200 μg/mL without activation, and 50, 100, 200, 300, 400, and 500 μg/mL with S9 activation. There was a concentration-related increase in mutant frequency in both the nonactivated and activated assays with a 4-hour exposure (mutant frequencies 100 mutants per 106 clonable cells over that of the solvent control). Toxicity in the cloned cultures (total growth of 50% of the solvent control) was observed at concentrations 100 μg/mL without activation and 300 μg/mL with activation.

The trifluorothymidine-resistant colonies for the cloned cultures and for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. There was an increase in the frequency of small colonies when the treated cultures were compared to the solvent control cultures. An increase in the frequency of small colonies is consistent with damage to multiple loci on chromosome 11 in addition to functional loss of the TK locus. Under the conditions of this study, the test article FTC/TDF was concluded to be positive in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.


Of the 3 compounds, only TDF had positive findings in genotoxicity studies (mouse lymphoma cell assay and UDS assay). The combination of FTC and TDF in a mouse lymphoma cell assay did not worsen the genotoxic potential of TDF. The combination of the 3 components is not expected to have an altered genotoxicity profile as compared with that of the individual agents.

2.6.6.5. Carcinogenicity

2.6.6.5.1. Emtricitabine: TP-0006: 2-Year Oral Oncogenicity Study in CD-1 Mice

This study was conducted to evaluate the oncogenic potential of FTC and to characterize any chronic toxicities in Charles River CD-1 mice following 104 weeks of daily oral gavage administration (Tabulated Summary 2.6.7.10.A, TOX-109). The main study consisted of 3 groups of 60 male and 60 female mice that received the test article at a dose level of 80, 250, or 750 mg/kg/day, and an additional group of 60 male and 60 female mice that served as the control and were administered the vehicle (0.5% aqueous methylcellulose in deionized distilled water). Three additional treatment groups each containing 40 animals per sex per group were designated for toxicokinetic evaluation at dose levels of 80, 250, and 750 mg/kg/day. In addition, a sentinel group (nondosed) containing 25 animals per sex were



used to evaluate fecal parasites and serology during the course of the study. The dose volume used in all groups was 10 mL/kg.

All animals were observed at least BID for morbidity, mortality, injury, overt clinical signs, and availability of food and water. Body weight and food consumption measurements were recorded for all animals during the course of the study. Detailed observations for clinical signs were recorded weekly for main study animals during the course of the study. Various hematologic evaluations were conducted at study termination for main study animals. Blood was collected for toxicokinetic evaluations at various time points during Weeks 2, 26, and 104. At study termination, complete necropsy examinations were performed and selected tissues were microscopically examined.

No effect of treatment was seen in any parameter evaluated, including survival, clinical and mass findings, body weight, food consumption, hematology, and macroscopic and microscopic pathology. No treatment-related microscopic changes suggestive of a carcinogenic effect were observed.

In summary, the daily administration of FTC for 2 years to Charles River CD-1 mice at dose levels at 80, 250, and 750 mg/kg/day did not produce any evidence of a carcinogenic effect.

Pharmacokinetic Evaluation

Emtricitabine was rapidly absorbed following all doses with peak plasma concentrations occurring at 0.5 to 1 hour postdose (see Tabulated Summary 2.6.5.4.A, TOX-109). AUC0 24hand Cmax increased proportionally with doses over the range of 80 to 750 mg/kg/day. In general, the exposures (AUC0 24h) in male mice were similar to those in female mice at all doses. AUC0 24h and Cmax values were higher at Week 26 compared to Week 2. At Week 104, FTC concentrations were below the limit of quantitation in most control animals. The average exposure in mice in this oncogenicity study ranged from 2.6- to 26.3-fold greater (at 80 and 750 mg/kg/day, respectively) than in humans receiving 200 mg/day.

2.6.6.5.2. Emtricitabine: TP-0006: 2-Year Oral Oncogenicity Study in CD Rats

This study was conducted to evaluate the oncogenic potential of FTC and to characterize any chronic toxicities in CD (Crl: CD® [SD]IGS BR) rats following 104 weeks of daily oral gavage administration (Tabulated Summary 2.6.7.10.B, TOX-108). The main study consisted of 3 groups of 60 male and 60 female rats that received the test article at dose levels of 60, 200, or 600 mg/kg/day, and an additional group of 60 male and 60 female rats that served as the control and were administered the vehicle (0.5% aqueous methylcellulose in deionized distilled water). Three additional treatment groups each containing 20 animals per sex per group were designated for toxicokinetic evaluation at dose levels of 60, 200, or 600 mg/kg/day. The dose volume was 10 mL/kg.



All animals were observed at least BID for morbidity, mortality, injury, overt clinical signs, and availability of food and water. Body weight and food consumption measurements were recorded for all animals during the course of the study. Detailed observations for clinical signs were recorded weekly for main study animals during the course of the study. Various hematologic evaluations were conducted at study termination for main study animals. Blood was collected for toxicokinetic evaluations at various time points during Weeks 2, 26, and 104. At study termination, complete necropsy examinations were performed and selected tissues were microscopically examined.

No effect of treatment was seen in any parameter evaluated including survival, clinical and mass findings, body weight, food consumption, hematology, and macroscopic and microscopic pathology. No treatment-related microscopic changes suggestive of a carcinogenic effect were observed.

In summary, the daily administration of FTC for 2 years to CD rats at dose levels of 60, 200, and 600 mg/kg/day did not produce any evidence of a carcinogenic effect.


Emtricitabine was rapidly absorbed following all doses, with peak plasma concentrations occurring at 0.5 hours postdose. AUC0 24h and Cmax results are summarized in Tabulated Summary 2.6.5.4.B (TOX-108). AUC0 24h and Cmax increased with dose over the range of 60 to 600 mg/kg/day. In general, exposures (AUC0 24h) in male rats were similar to those in female rats. AUC0 24h and Cmax values were higher at Week 26 compared to Week 2. The average exposure, based on Week 2 and Week 26 AUC0 24h values, in rats in this oncogenicity study ranged from 3.9- to 31.3-fold greater (at 60 and 600 mg/kg/day, respectively) than in humans receiving 200 mg/day.

2.6.6.5.3. Rilpivirine: 2-Year Carcinogenicity Study in Mice

TMC278 formulated in 0.5% m/v aqueous HPMC was administered once daily by oral gavage for 24 months (Tabulated Summary 2.6.7.10.C, TMC278-NC120). One control and 3 treated groups were given 0 (vehicle), 20, 60 or 160 mg/kg/day at a dose volume of 10 mL/kg. Each group consisted of 60 male and 60 female CD-1 mice (main animals) along with 24 male and 24 female satellite animals for toxicokinetic analysis (18 animals/sex in control group). The intended treatment duration was 104 weeks. Stop-dosing and termination rules communicated and agreed by FDA were applied due to increased mortality rates (Table 19).

Regular observations were made of mortality, clinical signs, palpable masses, body weight, and food consumption. Sampling for hematology was done at the end of the treatment period. A full necropsy was performed on all animals, including those found dead or sacrificed moribund during the study, and all gross lesions were recorded. A selection of organs was weighed. Tissues and organs indicated by protocol were sampled and preserved for histological examination. Samples of any abnormal tissues were also retained and processed for examination. In those cases where a lesion was not clearly delineated, contiguous tissue



was fixed with the grossly affected region and sectioned as appropriate. In addition to any masses, the lymph nodes draining the regions adjacent to those masses were also preserved.Blood samples were collected for toxicokinetic analysis on Day 1 and during Week 28.

The trend test for mortality with dose, when all treated groups were included, was statistically significant for male mice. Upon exclusion of the group treated with 160 mg/kg/day, the trend test was still significant. Pair-wise comparisons of the control group with the groups treated with 60 and 160 mg/kg/day were statistically significant. For females, the trend test for mortality with dose was not statistically significant. In male and female mice dosed with 160 mg/kg/day, hepatic tumors caused death more frequently than in males and females of the control group. Treatment-unrelated causes for mortalities included urogenital tract disease; glomerulonephritis and/or chronic nephropathy; malignant lymphoma; and ovarian cyst.

Table 19. Mortality in a Mouse Carcinogenicity Study with TMC278

0 20 60 160 Dose (mg/kg/day) M F M F M F M F

Initial number 60 60 60 60 60 60 60 60

Unscheduled deaths 25 40 35 37 39 30 42 43

Survival (%) 58 33 42 38 35 50 30 28

Week of treatment completed 104 102 104 102 104 102 100a 98

Week of termination 104 103 104 103 104 103 104 99

M: males, F: females, a: kept off dose until week 104.

There were no relevant changes in clinical signs. TMC278 did not affect the incidence or the time of appearance of palpable masses.

Treatment with TMC278 at doses up to 160 mg/kg/day was associated with a slightly higher body weight gain in all treated males and females at 60 and 160 mg/kg/day compared to control animals. Compared to controls, slightly higher food consumption in animals given 160 mg/kg/day and minimally higher food consumption in females at 60 mg/kg/day was evident.

No relevant changes in hematology were observed.

Gross lesions and organ weights indicated an increase in the incidence of liver masses in all groups treated with TMC278. In males and females treated with 160 mg/kg/day, the incidences of enlarged livers and pale areas in liver were also increased. Absolute and body weight-adjusted liver weight was increased (up to 3-fold) in males and females from all groups treated with TMC278, respectively.



In the liver, a statistically significant dose-related increase in total hepatocellular tumors (adenomas and carcinomas combined) was seen in males dosed with TMC278 from 20 mg/kg/day onwards. The dose-related increase in liver adenomas alone reached statistical significance in males treated at 60 and 160 mg/kg/day. An increase in the males with liver carcinomas that was not statistically significant was seen in all groups treated with TMC278 (see Table 20). The incidences of carcinomas and of adenomas for all groups treated with TMC278 and for the groups dosed with 60 and 160 mg/kg/day, respectively, were above the ranges expected from background data of the testing facility. The incidences increasing over background data are considered treatment related. The incidences of liver adenomas and of adenomas and carcinomas combined were statistically significantly increased and above the background data range in female mice treated with 60 and 160 mg/kg/day (Table 20).

Table 20. Neoplastic Liver Changes in a Mouse Carcinogenicity Study with TMC278

0 20 60 160 Dose (mg/kg/day) M F M F M F M F

Number of animals 60 60 60 60 60 60 60 60

Hepatocellular adenomas 8 1 14 0 21 8 19 16

Hepatocellular carcinomas 4 0 7 0 7 2 9 7


In addition, other treatment-related liver changes included eosinophilic, basophilic, and clear cell foci; centrilobular and generalized eosinophilic hypertrophy; and brown pigmented macrophages/Kupffer cells. A number of these changes were recorded at a higher incidence in males and females given 160 mg/kg/day and in females dosed with 60 mg/kg/day.

The degree and/or incidence of nephropathy/glomerulonephritis were slightly enhanced in females that received 160 mg/kg/day when compared with control females. These changes accounted for the granular appearance of kidneys noted at necropsy.

It was concluded that oral administration of TMC278 to CD-1 mice at doses of 20, 60, or 160 mg/kg/day produced a dose-related increase in total hepatocellular tumors (adenoma and carcinoma) in males dosed with 20 mg/kg/day and in males and females that received 60 and 160 mg/kg/day. For that reason, a NOAEL could not be determined. The associated systemic exposure expressed as Cmax and AUC values is presented in Table 21 below.



Table 21. Exposure to TMC278 (Cmax and AUC) in a Mouse Carcinogenicity Study with TMC278

Cmax (μg/Ml) AUC (μg.h/Ml)aDose

(mg/kg/day) Sampling Day M F M F

Day 1 9.9 13 59 61 20

Week 28 9.8 9.9 76 51

Day 1 23 24 239 182 60

Week 28 22 29 230 278

Day 1 41 38 440 345 160

Week 28 36 58 505 766


2.6.6.5.4. Rilpivirine: 2-Year Carcinogenicity Study in Rats

The design of the rat carcinogenicity study was in line with recommendations of the FDA Carcinogenicity Assessment Committee (CAC) (CAC recommendations: Facsimile of the feedback from the FDA on the carcinogenicity studies. 25 July 2005). The CAC requested a much higher top dose in the rat carcinogenicity study than tested in previous pivotal rat repeat-dose studies. For that reason, it was decided to include a fourth dose group in the carcinogenicity study to ensure that the study would finish with at least 3 dose groups in case the top dose would lead to increased mortality and early termination of that group.

TMC278 formulated in 0.5% m/v aqueous HPMC was administered once daily by oral gavage for 24 months (Tabulated Summary 2.6.7.10.D, TMC278-NC123). One control and 4 treated groups were given 0 (vehicle), 40, 200, 500, or 1500 mg/kg/day at a dose volume of 10 mL/kg. Each group consisted of 65 male and 65 female Sprague Dawley rats (main animals) along with 9 male and 9 female satellite animals for toxicokinetic analysis (6 animals/sex in the control group). The intended treatment duration was 104 weeks. Dosing of the females from Group 2 was completed in Week 98. All female groups were terminated in Week 99 due to increased mortality rates according to stop-dosing and termination rules in agreement with the FDA.



Regular observations were made of mortality, clinical signs, body weight, and food consumption. The onset and progression of any palpable masses were evaluated after 6 months of treatment. Ophthalmic examinations were carried out in Weeks 26 and 52, and prior to termination. Samples for clinical laboratory investigations (hematology, coagulation, blood chemistry, and urinalysis) were taken after 12 and 24 months of dosing in the surviving males and females. A full necropsy was performed on all main animals, including those found dead or sacrificed moribund during the study, and all gross lesions were recorded. A selection of organs was weighed. Tissues and organs indicated by protocol were sampled and preserved for histological examination. Samples of any abnormal tissues were also retained and processed for examination. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate. In addition to any masses, the lymph nodes draining the regions adjacent to those masses were also preserved. Blood samples for toxicokinetic analysis were taken on the first day of dosing and after 27 and 39 weeks of dosing.

There was no effect of treatment on mortality (see Table 22). No treatment-related pathologies contributing to death and no adverse clinical signs were observed.

Body weight, food consumption, and food conversion efficiency were not affected by treatment and the ophthalmic examination did not reveal any treatment-related lesions.

The hematological investigations did not indicate any changes in the peripheral blood that were related to the administration of TMC278, in particular there was no evidence of any treatment-related leukemias.

Table 22. Mortality in the Rat Carcinogenicity Study with TMC278

0 40 200 500 1500 Dose (mg/kg/day) M F M F M F M F M F

Initial number 65 65 65 65 65 65 65 65 65 65

Unscheduled deaths 40 45 40 46 38 35 37 33 44 34

Survival (%) 39 31 39 29 42 46 43 49 32 48

Week of treatment completed 104 99 104 98 104 99 104 99 104 99

Week of termination 104 99 104 99 104 99 104 99 104 99




Serum chemistry samples at Week 52 showed increased activities of ALP, ALT, AST, and GGT and increased concentration of creatinine and to a lesser extent of urea in males. Moreover, ALP activity in females and the albumin concentration and A/G ratio in males and females were higher than the respective control values. Serum concentrations of 2-globulin in males; 1-globulin in females; and cholesterol, triglycerides, and -globulin in males and females were lower that the corresponding control values. The effects generally attained statistical significance at doses of 500 and 1500 mg/kg/day. The effects on creatinine, triglycerides, the globulins, and the A/G ratio showed a statistically significant difference from controls from a dose of 200 mg/kg/day, and above. The lowering of 2-globulin was already statistically significant at a dose of 40 mg/kg/day.

At termination, ALP activity and glucose concentration were elevated in males; potassium in females; and creatinine in males and females. Cholesterol and -globulin concentrations were reduced in males, as were the triglycerides concentrations in males and females. The effects generally attained statistical significance at doses of 500 and 1500 mg/kg/day. The effect on cholesterol was statistically significant from a dose of 200 mg/kg/day, and above, and the decrease of -globulin was statistically significantly different from control at a dose of 40 mg/kg/day.

Urinalysis showed lower potassium concentrations in females throughout the study and in males at termination. The effect in females attained statistical significance in the groups given 500 and 1500 mg/kg/day, whereas that in males was statistically significant from a dose of 200 mg/kg/day, and above. Chloride concentration in urine was strongly elevated in a dose-related fashion from 150% of the control value in females that received 200 mg/kg/day up to a more than a 4-fold increase in the males and females dosed with 1500 mg/kg/day. The effect in females was more prominent than in males. Urinary pH was significantly lower in the group given 1500 mg/kg/day (in females throughout the dosing period and in males at termination). Urinary protein was significantly higher in females dosed with 1500 mg/kg/day at Week 52.

At necropsy, white or pale contents of the GI tract were recorded in animals dosed with 200 mg/kg/day and higher doses. This observation is related to the color of the formulation and is associated with the recording of pale feces at clinical observations. A minimal increase in thyroid gland weight was recorded in high dose animals. Increased incidences of masses were noted in the liver of all groups of females dosed with TMC278 and in thyroid gland of males that received TMC278, in a non-dose-related fashion. No effects were noted on organ weights, except for a statistically significant increase of the absolute weight of the thyroid plus the parathyroid glands in males and females dosed with 1500 mg/kg/day.



In liver, an increase in hepatocellular adenomas was seen in males and females given 200, 500, or 1500 mg/kg/day, and also in females given 40 mg/kg/day. There was no apparent dose-related trend and the differences from control values did not reach statistical significance. When compared with the historical background data, the incidence of tumors in females was above the background level in all groups treated with TMC278; whereas in the males, the incidence of tumors was equal to the maximum historical background incidence recorded. These changes are considered to be related to the administration of TMC278. There were no nonneoplastic findings associated with the liver tumors.

In thyroid gland, when compared with the controls, a statistically significant increase of the number of follicular cell adenomas and of adenomas and carcinomas combined was seen in all groups of male and female rats treated with TMC278, with only a marginally apparent dose-related trend. The incidence of follicular cell adenomas was above background levels in the males and females at 200, 500, or 1500 mg/kg/day. The incidence of the carcinomas was above background levels in males only at 200 and 1500 mg/kg/day. These neoplastic changes presented in Table 23 are considered to be treatment related. Nonneoplastic changes related to these tumors included increased incidences of follicular cell hypertrophy and cystic follicular cell hyperplasia in females of all groups treated with TMC278. In males dosed with 200, 500, or 1500 mg/kg/day, the incidence of follicular cell hypertrophy was increased.

None of the liver or thyroid gland tumors was considered contributory to death in any rat.

Table 23. Neoplastic Liver and Thyroid gland Changes in a Rat Carcinogenicity Study with TMC278

0 40 200 500 1500 Dose (mg/kg/day) M F M F M F M F M F

Initial number 65 65 65 65 65 65 65 65 65 65

Liver

Hepatocellular adenomas 0 1 1 5 3 5 3 6 3 4

Thyroid gland

Follicular cell adenoma 1 0 3 0 4 3 5 3 5 4

Follicular cell carcinoma 0 0 0 1 2 1 1 0 2 1


Inflammatory exudate in the nasal passages, with associated changes in nasal mucosa (including hyperplasia and/or inflammatory cell infiltration of the respiratory epithelium and respiratory metaplasia of the olfactory epithelium), were seen in males and females given 1500 mg/kg/day. When compared with the controls, there was an increased incidence and/or severity of foamy alveolar macrophages (and associated alveolar epithelial hyperplasia) in the lungs of females given 200, 500, or 1500 mg/kg/day. In the liver, there was an increased incidence of sinusoidal dilatation in females given 1500 mg/kg/day.



It was concluded that the oral administration of TMC278 to Sprague Dawley rats at doses of 40, 200, 500, or 1500 mg/kg/day produced neoplastic changes in liver and thyroid gland. Increased incidences of liver adenomas were seen in males and females given 200 mg/kg/day or above and in females at 40 mg/kg/day without attaining statistical significance. Moreover, a statistically significant increase in follicular cell adenoma and/or carcinoma was seen in the thyroid gland among all groups of males and females treated with TMC278, generally without a dose-related trend. For that reason, a NOAEL could not be determined. The associated systemic exposure expressed as Cmax and AUC values is presented in Table 24 below.

Table 24. Exposure to TMC278 (Cmax and AUC Values) in a Rat Carcinogenicity Study with TMC278

Cmax (μg/mL) AUC (μg.h/mL)a

Dose (mg/kg/day) Sampling Day M F M F

Day 1 1.6 2.2 19 17

Week 27 0.85 2.2 4.5 15 40

Week 39 0.82 2.1 6.3 14

Day 1 2.6 4.2 34 40 b

Week 27 1.2 5.2 11 36 200

Week 39 1.3 4.7 8.2 41

Day 1 4.5 6.5 45 63

Week 27 2.0 5.7 14 70 500

Week 39 1.8 8.5 14 46

Day 1 6.1 7.0 58 81

Week 27 2.7 10. 20 81 1500

Week 39 2.2 9.4 18 84

a AUCinf: on Day 1 otherwise AUC0 24h, M: males, F: females; b: AUC0 24h

2.6.6.5.5. Tenofovir DF: An Oral Carcinogenicity Study of Tenofovir DF in the Albino Mouse

In a carcinogenicity study of TDF, Swiss Crl:CD1(ICR) BR mice (60/sex/group) received daily doses of 0, 100, 300, or 600 mg/kg/day by oral gavage for 104 weeks (Tabulated Summary 2.6.7.10.E, M990205).



The following parameters were measured during the study: mortality; signs of ill health and reaction to treatment (BID); detailed physical examination (weekly); the presence of palpable masses (from Week 26 onwards); body weights and food consumption (weekly); ophthalmology (once pretreatment, Week 52, and Week 104); and hematology (prior to commencement of treatment on health screen animals). Blood smears were obtained from all animals at 12, 18, and 24 months. In the absence of any treatment-related effect, only the control and high level smears were evaluated. Toxicokinetic sampling was performed on Days 1 and 180 at predose, 5 minutes (Day 1 only), 15 minutes, 30 minutes, and at 1, 2, 4, 8, 12, and 24 hours postdose on 3 animals/sex/group/time point (high dose group only). Gross pathology (necropsy) was performed on all main group and toxicokinetic animals. Histological examination was performed on all tissues from main animals and the results were peer reviewed.

Treatment-related clinical signs were restricted to salivation. Salivation following dose administration was noted for all animals receiving 300 or 600 mg/kg/day, with a higher incidence being seen in animals receiving 600 mg/kg/day.

A reduced group mean body weight was recorded for animals receiving 300 (4.2% and 0.6% for males and females, respectively) or 600 mg/kg/day (4.6% and 3.3% for males and females, respectively) when compared to the concurrent controls over the 104-week treatment period. The differences from the controls were dosage related and on occasion attained statistical significance. There were no effects on body weight in animals receiving 100 mg/kg/day.

There were no treatment-related effects on food consumption, hematology parameters, or ophthalmoscopy.

Treatment-related effects were observed in the bone marrow, duodenum, kidneys, liver, and testes. The results of the histopathological examination revealed a low incidence of treatment-related lesions in the duodenum (adenoma/adenocarcinoma) at 600 mg/kg/day. Nonneoplastic lesions were observed in duodenum (epithelial hyperplasia) at 100, 300, and 600 mg/kg/day; in liver (eosinophilic and basophilic cell foci, hepatocellular hypertrophy, nuclear inclusions, cyto/karyomegaly, single cell necrosis, proliferation of sinusoidal cells) and testes (vascular mineralization) at 300 or 600 mg/kg/day; and in kidneys (karyomegaly) and bone marrow (hypercellularity: myeloid) at 600 mg/kg/day. A NOEL for nonneoplastic lesions (by histological criteria) was not established in this study.

In conclusion, lifetime daily administration of TDF at dosage levels of up to 300 mg/kg/day given for 104 weeks did not reveal any evidence of carcinogenicity in Swiss Crl:CDR-1(ICR)BR mice. At 600 mg/kg/day, a low incidence of duodenal tumors, possibly treatment related, was observed.




Concentrations of TFV in plasma were determined during a 2-year carcinogenicity study of oral TDF in male and female albino mice (Tabulated Summary 2.6.5.4.AA, M990205). Blood samples were taken from mice (3 mice/sex/time point) on Day 1 and Day 180 following daily oral dosing of 600 mg/kg/day TDF. Time points included predose; 5 (Day 1 only), 15, and 30 minutes; and 1, 2, 4, 8 (on Day 180, 2 males and 2 females were used for sampling), 12 and 24 hours postdose. Samples were processed for plasma and concentrations of TFV in plasma were determined using a validated LC/MS method. Pharmacokinetic parameters for TFV in plasma were determined by noncompartmental methods. Following the first oral dose in male and female mice, concentrations of TFV reached mean peak values of 11.5 and 17.6 mg/mL, respectively. The corresponding calculated mean AUCinf values ( g·h/mL) in male and female mice were 50.4 and 48.7, respectively. The extent of exposure (AUC0 24h) of TFV appeared to be similar between the male and female mice on Days 1 and 180. AUCinf on Day 1 appeared to be comparable to AUC0 24h on Day 180 in both the male and female mice, suggesting TFV possesses linear toxicokinetic properties at a dose of 600 mg/kg/day.

2.6.6.5.6. Tenofovir DF: An Oral Carcinogenicity Study of Tenofovir DF in the Albino Rat

In a carcinogenicity study of TDF, Sprague-Dawley CD rats (60/sex/group) received daily doses of 0, 30, 100, or 300 mg/kg/day by oral gavage for 103/104 weeks (Tabulated Summary 2.6.7.10.F, R990204). The high dose animals (300 mg/kg/day) were initially assigned as the low dose group and received 10 mg/kg/day until Day 10 when the dose was increased to 300 mg/kg/day. Toxicity was assessed by clinical observations (BID); body weight and food consumption (weekly); ophthalmologic examinations (pretreatment, Weeks 52 and 104); hematology (Months 12, 18, and at terminal necropsy); gross findings and histopathology. Toxicokinetics were determined in satellite animals to assess exposure on Days 1 and 180.

Survival ranged from 27% to 47% at termination, which is within the historical range for the laboratory. Treatment-related clinical signs included salivation in the 100 and 300 mg/kg/day animals. The number of rats with clinically observed masses was similar between control and treated groups. Lower mean body weights were recorded for males (100 and 300 mg/kg/day) and females (300 mg/kg/day) during the first 3 quarters of the study (with differences reaching statistical significance on occasion). Mean food consumption for all treated groups was comparable to controls. There were no changes in hematology (RBC, WBC) at Months 12, 18, or 24.

A dose-related increase in the incidence of renal tubular karyomegaly was observed in treated animals. This finding is consistent with previous observations in subchronic and chronic studies of TDF in rats.

Once daily oral administration of TDF to rats for 103/104 consecutive weeks did not result in any increase in the incidence of neoplastic lesions in various organ and tissue systems



examined. The benign and malignant tumors observed followed no pattern, appeared to be spontaneous in nature, and were within the limits reported for aging CD Sprague-Dawley rats. Using toxicokinetic data from this study, the safety margin based on plasma AUCss is approximately 5x (mean AUCss were 16.7 and 15.7 g·h/mL in male and female rats, respectively; human AUCss is 3 g·h/mL).

In conclusion, lifetime daily administration of TDF at dosage levels up to 300 mg/kg/day did not reveal any evidence of carcinogenicity in Sprague-Dawley rats.


Concentrations of TFV in plasma were obtained during a 2-year carcinogenicity study of oral TDF in male and female albino rats (Tabulated Summary 2.6.5.4.EE, R990204). The study involved taking blood samples from rats (3 rats/sex/time point) on Day 1 and Day 180 following daily oral dosing of 30, 100, and 300 mg/kg TDF. Time points included predose and 0.25, 0.5, 1, 2, 4, 8, and 24 hours postdose. Samples were processed for plasma and concentrations of TFV in plasma were determined using a validated LC/MS method. Pharmacokinetic parameters for TFV in plasma were determined by noncompartmental methods. Following the first oral dose in male rats, concentrations of TFV in plasma reached mean peak values of 0.54, 1.31, and 1.99 g/mL at the 30, 100, and 300 mg/kg/day dose levels, respectively. The corresponding calculated mean AUCinf values in male rats were 2.53, 5.82, and 19.36 g·h/mL. For female rats, the Cmax following the first dose was 0.77, 1.42, and 6.61 g/mL at the 30, 100, and 300 mg/kg/day dose levels, respectively. The corresponding calculated mean AUCinf values in female rats were 2.39, 5.27, and 14.18 g·h/mL. On Day 180, mean plasma AUCss(0 24h) at dose levels of 30, 100, and 300 mg/kg/day were 2.70, 6.60, and 16.69 g·h/mL, respectively, for male rats; and 3.06, 7.46, and 15.72 g·h/mL, respectively, for female rats. The extent of exposure (AUC0 24h) of TFV appeared to be similar between the male and female rats on Days 1 and 180. Cmax values were generally comparable between males and females. Exposure was linear within each dose level, but did not increase in a dose-proportional fashion when doses were increased from 30 to 300 mg/kg/day.


Emtricitabine and TDF have both demonstrated low carcinogenic potential in conventional 2-year bioassays at exposures that exceeded (TDF) or far exceeded (FTC) human exposures at the therapeutic dose. It is considered unlikely that combination dosing would change this profile as no exposure difference would be expected and no exacerbation of toxicity/genotoxicity is expected.

The carcinogenicity studies with TMC278 in mice and rats demonstrated in both species an increased incidence of hepatocellular tumors (adenomas and carcinomas in mice and adenomas in rats). In the latter species, also follicular cell adenomas and carcinomas in the thyroid gland were noted. No neoplastic lesions were noted associated with the other targets of toxicity of TMC278. The carcinogenic potential of TMC278 is discussed in Section 2.6.6.9.8.



Given the difficulty of extrapolating rodent results to humans and the large number of animals required to carry out these studies, the conduct of carcinogenicity studies with the FTC/RPV/TDF combination seems unjustifiable.

2.6.6.6. Reproductive and Developmental Toxicity

2.6.6.6.1. Fertility and Early Embryonic Development

2.6.6.6.1.1. Emtricitabine: Study of Fertility and Early Embryonic Development of TP-0006 Administered by Gavage to CD-1 Mice (Segment I)

Four groups of 20 to 21 male and 20 female mice received vehicle or 250, 500, or 1000 mg/kg/day of FTC by oral gavage (Tabulated Summary 2.6.7.12.A, TOX036). The dose was administered as 2 equal installments given approximately 6 hours apart. Male mice were dosed 28 days prior to breeding, 21 days during mating, and 21 days postmating for a total of 70 days. Females were dosed 14 days prior to breeding, 21 days during mating, and 7 days during gestation for a total of 42 days. There were no treatment-related deaths and all mice were observed to be normal during all observation periods. Body weight, body weight change, and food consumption were also unaffected by treatment. There was no indication of reproductive toxicity. Mating and fertility indices were unaffected by treatment, as was epididymal sperm number and motility. There were no gross pathological findings at necropsy. Embryonic development was also unaffected by treatment with FTC; there was no effect on the number of ovarian corpora lutea or uterine implantation sites per dam; the number or percent of resorptions and nonlive implants per litter; the number and percent of litters with nonlive implants; the number of live implants per litter; the number or percent of litters with resorptions; or the percent preimplantation or postimplantation loss per litter. In this study, the NOAEL of FTC for male and female mouse systemic, reproductive, and developmental toxicity was considered to exceed 1000 mg/kg/day.

2.6.6.6.1.2. Emtricitabine: A Fertility Study in Male Rats Given 524W91 by Gavage

The potential for adverse effects of orally administered FTC on the reproductive performance of male Sprague-Dawley rats was evaluated (Tabulated Summary 2.6.7.12.B, TTEP/95/0028). Male rats were given once daily doses at 0, 150, 750, or 3000 mg/kg/day for 73 days prior to mating and throughout the mating period. Body weight, feed consumption, organ weights, gross lesions, histopathology of reproductive organs, sperm parameters, and fertility were evaluated. Naive females were cohabited with treated males and monitored for evidence of mating. On Gestation Day 13 (GD 13), the females were inspected for pregnancy by cesarean section. The number of viable fetuses, number early and late resorptions, and number of deaths were then quantified. The only treatment-related effect was salivation immediately following dosing at 3000 mg/kg/day. There were no adverse effects on fertility, reproductive performance, sperm count or motility, and there were no pathological changes in the male reproductive organs. Based on these findings, the NOEL was 750 mg/kg/day.



2.6.6.6.1.3. Rilpivirine: Female Rat Fertility Study and Early Embryonic Development Study

TMC278 suspended in 0.5% (m/v) aqueous HPMC was administered once daily, by oral gavage, to groups of 24 female Sprague Dawley rats at 0 (vehicle), 40, 120, or 400 mg/kg/day in a dose volume of 10 mL/kg (Tabulated Summary 2.6.7.12.D, TMC278-NC125). Treatment started 2 weeks prior to mating, during mating, and until Day 7 of pregnancy. The females were paired 1:1 for mating with untreated males. Successful mating was established on the basis of sperm detection in vaginal smears. Regular observations were made for clinical signs, morbidity, and mortality. Female body weights were determined weekly until pregnancy, and on pregnancy Days 0 (day of sperm detection), 8, and 14. Food consumption was determined weekly during the premating period and pregnancy. Estrous cycle was determined on vaginal smears taken daily during the mating period and the precoital interval was calculated. The females were sacrificed on Day 14 of pregnancy for evaluation of pregnancy status and determination of copulation and fertility rates (e.g., number of corpora lutea, implantations, and live fetuses).

No mortalities were noted. Moreover, there were no relevant clinical signs and no relevant effects on body weight, food consumption, gross pathology, estrous cycle, mating or pregnancy rate, the number of corpora lutea, implantations or live fetuses, or early embryonic development indices. It was concluded that there was no effect on female fertility, fecundity, or early embryonic development up to 400 mg/kg.

Therefore, the NOAEL for female fertility, fecundity, and early embryonic development was at least 400 mg/kg/day.

2.6.6.6.1.4. Rilpivirine: Male Rat Fertility Study

TMC278 suspended in 0.5% (m/v) aqueous HPMC was administered once daily, by oral gavage, to groups of 25 male Sprague Dawley rats at 0 (vehicle), 100, 400, or 1600 mg/kg/day in a dose volume of 10 mL/kg (Tabulated Summary 2.6.7.12.C, TMC278-NC124). Treatment started 10 weeks prior to mating, during mating, and for 3 to 4 weeks after the mating period. The males were paired 1:1 for mating with untreated females. Successful mating was established on the basis of sperm detection in vaginal lavage. Regular observations were made for clinical signs, morbidity, and mortality. Male body weight was recorded weekly throughout the dosing period and food consumption during the premating period. At necropsy of the males, 3 to 4 weeks after the end of the mating period, gross lesions were recorded. The testes and epididymides were removed, weighed, and examined histopathologically. One of the cauda epididymides from each male was sampled for sperm motility, concentration, and morphology. The liver and thyroid gland were weighed and fixated.

There were no mortalities associated with TMC278. All pairs except one (male dosed with 100 mg/kg/day) mated within 4 days after pairing. There were no relevant clinical signs and no relevant effects on body weight, food consumption, gross or histopathological lesions, weights of epididymides or testes; and no adverse effects on the motility, concentration, or



morphology of the sperm. There was no effect on fertility up to 1600 mg/kg/day. Weights of liver and thyroid gland showed a dose-related increase (absolute and/or relative to body weight) in all groups receiving TMC278.

The NOAEL for male fertility is at least 1600 mg/kg/day.

2.6.6.6.1.5. Tenofovir DF: Oral (Gavage) Fertility and General Reproduction Toxicity Study of GS–4331–05 (bis-POC PMPA) in Sprague-Dawley Rats

Two-hundred Crl:CD®BR VAF/Plus® (Sprague-Dawley) rats were randomly assigned to 4 dosage groups (Groups I through IV; 25 rats per sex, per group) (Tabulated Summary 2.6.7.12.E, 98-TOX-4331-006). The test article, TDF (100, 300, or 600 mg/kg/day) or the vehicle (suspension vehicle), were administered orally (via gavage) to the male rats once daily beginning 28 days before cohabitation (maximum 21 days) and continuing through the day before sacrifice, and to female rats once daily beginning 15 days before cohabitation (maximum of 21 days) and continuing through presumed GD 7. The dosage volume was 10 mL/kg, adjusted daily for body weight changes, and given at approximately the same time each day. Rats were observed for viability twice each day of the study. These rats were also examined for clinical observations and general appearance weekly during acclimation. Observations for clinical signs of effects of the test article, abortions, premature deliveries, and deaths were also made daily before and approximately 30 5 minutes postdose through Day 7 of the study; beginning on Day 8 of the study, these observations were made 60 5 minutes after administration. These observations were also recorded once daily during the postdosage period. Body weights were recorded weekly during acclimation, daily during the dosage and postdosage periods, and at sacrifice. Feed consumption values for male rats were recorded weekly throughout the study, except during cohabitation. Feed consumption values for female rats were recorded weekly to cohabitation and on GDs 0, 7, 8, 12, 14, 18, and 20. After completion of the cohabitation period, male rats were sacrificed and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. Tissues with gross lesions were preserved for possible future evaluation. The following organs were weighed: right testis, left testis, left epididymis (whole and cauda), right epididymis, seminal vesicles (with and without fluid), and prostate and kidneys (paired weight). A portion of the left cauda epididymis was used for evaluation of cauda epididymal sperm count, concentration, and motility using computer-assisted sperm analysis (CASA). Female rats were sacrificed on GD 20 or an estimated GD 20, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. Ovaries and tissues with gross lesions were retained and the kidneys were excised, weighed (paired weight), and retained for possible histologic examination. The number of corpora lutea in each ovary was recorded. The uterus of each rat was excised and examined for pregnancy; number and distribution of corpora lutea; implantations; live and dead fetuses; and early and late resorptions. Fetuses were weighed and examined for sex and for gross external alterations. Approximately one-half of the fetuses in each litter were fixed in Bouin’s solution; the remaining fetuses were retained in alcohol for possible further evaluation. Rats that died or were sacrificed because of moribund condition were examined for the cause of death or moribund condition on the day the observation was made. The rats were examined for gross lesions. Testes and epididymides of the 1 male rat that died were excised and individual reproductive organ and



paired kidney weights were recorded. The epididymides and gross lesions were retained. Pregnancy status and uterine contents of female rats were recorded. Ovaries were retained and the kidneys were excised, weighed (paired weight), and retained for possible examination.

For male rats, no deaths were attributed to administration of TDF. The death of 1 rat in the 300-mg/kg/day dosage group on Day 54 of study was the result of an intubation accident. All other male rats survived until scheduled sacrifice. Significantly increased numbers of rats in the 100, 300, and 600 mg/kg/day dosage groups had excess salivation, and significantly increased numbers of rats in the 600-mg/kg/day dosage group had a dry, red perioral substance. No necropsy observations were considered test article related. The weight of the paired kidneys and the ratios of the kidney weight to the terminal body weight were reduced and/or significantly reduced in the 300- and 600-mg/kg/day dosage groups. Body weight gains were significantly reduced in the 600-mg/kg/day dosage group on Days 1 to 8 of the study. This reduction resulted in the significantly reduced average body weight on Day 8 of the study in this dosage group. Body weights were then significantly increased on Days 8 to 15 of the study in the 600-mg/kg/day dosage group. After Day 15 of the study, body weight gains were comparable among the 4 dosage groups. Reflecting the reduced body weight gains on Days 1 to 8 of the study, absolute (g/day) and relative (g/kg/day) feed consumption values were significantly reduced on Days 1 to 8 of the study. Relative feed consumption values were significantly increased in the 600-mg/kg/day dosage group at most tabulated intervals after Day 8 of the study. All mating and fertility parameters were unaffected by dosages of the test article as high as 600 mg/kg/day.

For female rats, 2 rats in the 300-mg/kg/day group and 1 rat in the 600 mg/kg/day group, were moribund sacrificed, and 1 rat in the 600-mg/kg/day dosage group was found dead. These moribund sacrifices and deaths were not considered test article related because: (1) they occurred as a result of an intubation error or injury, and (2) no test article-related deaths occurred in the male rats administered these dosages. All other female rats survived until scheduled sacrifice. Significantly increased numbers of rats in the 600-mg/kg/day dosage group had a red or brown dry perioral substance, excess salivation, and urine-stained abdominal fur during the precohabitation period. A red or brown dry perioral substance and excess salivation occurred in 2 or 3 rats in the 300-mg/kg/day dosage group during the precohabitation period and in 2 or 3 rats in the 300- and 600-mg/kg/day dosage groups during gestation. No necropsy observations were considered test article related. Over the entire precohabitation period (Days 1 to 15 of the study), body weight gains were comparable and did not differ significantly among the dosage groups. However, significant body weight loss occurred in the 600-mg/kg/day dosage group from Days 1 to 8 of the study, resulting in significantly reduced average body weights on Days 5 through 8 of the study. Reflecting the reduced body weight gains on Days 1 to 8 of the study, absolute (g/day) and relative (g/kg/day) feed consumption values were significantly reduced in the 600-mg/kg/day dosage group on Days 1 to 8 and 1 to 15 of the study. Body weights and body weight gains during gestation were unaffected by dosages of the test article as high as 600 mg/kg/day. Absolute and relative feed consumption values in the 300- and 600-mg/kg/day dosage groups were significantly increased at several tabulated intervals after DG 8. The number of rats with 6 or more consecutive days of distress during the precohabitation dosage period was



significantly increased, resulting in a significant decrease in the number of estrous stages per 14 days in the 600-mg/kg/day dosage group. No cesarean-sectioning or litter parameters were affected by dosages of the test article as high as 600 mg/kg/day. No gross external alterations (malformations or variations) were caused by dosages of the test article as high as 600 mg/kg/day.

On the basis of these data, the NOEL for TDF for the male rats is less than 100 mg/kg/day (the 100 mg/kg/day and higher dosages caused adverse clinical observations; the 300 and 600 mg/kg/day dosages caused reductions in kidney weights; and the 600 mg/kg/day dosage caused reductions in body weight gain and feed consumption). The NOEL for the female rats was 100 mg/kg/day (the 300 and 600 mg/kg/day dosages caused adverse clinical observations and a compensatory increase in feed consumption values after the completion of the dosage period; the 600 mg/kg/day dosage also caused body weight loss and reduced feed consumption values in the first week of dosage). The reproductive NOEL is 300 mg/kg/day (the 600 mg/kg/day dosage altered estrous cycling in the female rats; however, there was no effect on mating or fertility). The developmental NOEL is greater than 600 mg/kg/day (no effects on cesarean-sectioning or litter parameters occurred at the highest dosage tested).

2.6.6.6.2. Embryo-Fetal Development

2.6.6.6.2.1. Emtricitabine: A Dose Range-Finding Study of the Effects of TP-0006 on Embryo/Fetal Development in Mice

A dose range-finding study was conducted to study the potential maternal and developmental toxicity of FTC in CD-1 mice for the purpose of selecting doses for the definitive study (Tabulated Summary 2.6.7.11.A, TOX033). On GDs 6 to 15, FTC was orally administered at 0, 250, 500, and 1000 mg/kg/day as a divided dose with 6 hours between doses (0.5% methylcellulose vehicle). On GD 18, laparohysterectomies were performed on all females and standard endpoints were measured. One high-dose female delivered 2 pups of 13 total on GD 18, just prior to the scheduled necropsy. All other animals progressed normally to the GD 18 necropsy. There were no treatment-related internal findings at any dose level, including gravid uterine weight, numbers of fetuses, fetus weights, sex ratios, external developmental malformations and variations, early and late resorptions, total implantations, and corpora lutea numbers. Maternal body weight gain and feed consumption were unaffected at all dose levels. Based upon these results, doses of 250, 500, and 1000 mg/kg/day were selected for the definitive mouse teratology study.



2.6.6.6.2.2. Emtricitabine: A Study of the Effects of TP-0006 (FTC) on Embryo/Fetal Development in Mice

The potential for FTC to cause developmental and maternal toxicity was evaluated in this GLP-compliant study (Tabulated Summary 2.6.7.13.A, TOX037). Groups of 25 pregnant mice were orally administered FTC suspended in 0.5% aqueous methylcellulose or vehicle alone, given BID as a divided dose with 6 hours between doses, from GDs 6 through 15. Doses tested were 0, 250, 500, and 1000 mg/kg/day (5 mL/kg/dose; 10 mL/kg/day). Body weights and feed consumption were recorded at the specified intervals. On GD 18, laparohysterectomies were performed on all females and data for maternal and fetal indices collected. Dams in satellite groups for each dose level were treated similarly, except that on GD 15 following a single dose of 250, 500, or 1000 mg/kg, they were exsanguinated at 0.5 hour postdose and plasma was harvested for toxicokinetic evaluations. All maternal animals survived to the necropsy on GD 18; there were no treatment-related maternal findings at that time. Feed and body weight data were all normal. Intrauterine growth and survival of fetuses were not affected by FTC at any dose level, and there were no external, visceral, or skeletal malformations or developmental variations observed at any dose. Toxicokinetic evaluations demonstrated dose proportional exposures to the drug with mean plasma values at 0.5 hour postdose of 52 15, 88 14, and 186 14 g/mL for the low-, mid-, and high-dose groups, respectively. Based on the results of this study, a dose level of 1000 mg/kg/day was considered to be the NOAEL for maternal and developmental toxicity of FTC when administered BID to mice.

2.6.6.6.2.3. Emtricitabine: A Dose Range-Finding Study of the Effects of TP-0006 on Embryo/Fetal Development in Rabbits

A GLP-compliant dose range-finding study of TP-0006 (FTC) for a developmental toxicity study was conducted in New Zealand White rabbits (Tabulated Summary 2.6.7.11.A, TOX034). Groups of 5 female artificially inseminated rabbits were dosed with FTC in 0.5% methylcellulose at 0, 250, 500, and 1000 mg/kg/day, administered in two equally divided doses with 6 hours between doses (GDs 7 to 19; GD 0 = day of insemination). Standard data parameters (body weights, feed consumption) were collected throughout the study. On GD 29, a laparohysterectomy was performed and the uteri and their contents examined. All females survived to the scheduled necropsy with no evidence of toxic or pharmacologic effects. Mean maternal body weight gain and feed consumption were normal. Growth and survival of the conceptuses were also normal. There were no external malformations or variations observed in fetuses at any dose level.

2.6.6.6.2.4. Emtricitabine: A Study of the Effects of TP-0006 on Embryo/Fetal Development in Rabbits

The potential for FTC to cause developmental and maternal toxicity were evaluated in rabbits (Tabulated Summary 2.6.7.13.B, TOX038). Groups of 20 artificially inseminated rabbits were orally dosed with FTC in 0.5% aqueous methylcellulose from GDs 7 through 19. Dose levels were 0, 100, 300, and 1000 mg/kg/day. Dosing occurred BID, with approximately 6 hours between doses, at a dose volume of 5 mL/kg/dose (10 mL/kg/day). Clinical



observations for signs of toxicity, body weights, and feed consumption were recorded. On GD 29, a laparohysterectomy was performed on all rabbits and the uteri and ovaries examined. Standard fetal evaluations were made to evaluate any effects on external, visceral, or skeletal development. Plasma concentrations of drug in maternal and fetal circulation was evaluated in a satellite group of rabbits (n = 5/group) on GDs 19 (maternal exposure) and 20 (fetal/maternal exposure). There were no test article-related deaths or abortions. The only test article-related clinical observation was decreased defecation in the high-dose group. Body weight gain and feed consumption were reduced in the 300 and 1000 mg/kg/day groups during GDs 11 to 15 (high dose only), 15 to 18, and/or 18 to 20. Intrauterine growth and survival were unaffected by FTC, and there were no treatment-related developmental malformations or variations in fetuses at any dose.

Toxicokinetic evaluation demonstrated that FTC was rapidly absorbed with Cmax occurring generally within 1 hour postdose. AUC and Cmax increased linearly with dose. Plasma elimination t1/2 was 3 to 4 hours at all dose levels. Mean maternal exposures (AUC0 24h) on GD 19 were 87, 315, 1258 g.h/mL for the low-, mid-, and high-dose groups, respectively. Fetal/maternal exposure ratios were around 0.4 to 0.5 at 1 hour after dosing (at Tmax) for all dose levels.

Based on the results of this study, a dose level of 100 mg/kg/day was considered to be the NOAEL for maternal toxicity and a dose level of 1000 mg/kg/day was considered the NOAEL for developmental toxicity of FTC when administered BID to rabbits.

2.6.6.6.2.5. Rilpivirine: Embryo-Fetal Development Studies in Rats (with TMC278 Base)

The doses for the rat embryo-fetal development study were selected on the basis of the results of a pilot study (Tabulated Summary 2.6.7.11.B, TMC278-NC127) in which no significant effects occurred. TMC278 base dissolved in PEG400 + CA was administered once daily, by oral gavage, from GDs 6 to 17 to groups of 30 (6 satellite animals for toxicokinetic sampling) pregnant Sprague Dawley rats at 0 (vehicle, 24 animals), 40, 120, or 400 mg/kg/day at a dose volume of 10 mL/kg (Tabulated Summary 2.6.7.13.C, TMC278-NC105). Regular observations were made for clinical signs, morbidity, mortality, body weight, and food intake. Blood samples from satellite animals (3 time points over a 24-hour period from 3 females per group) were collected on GDs 6 and 16. The satellite animals were killed after the last sampling. The dams from the main study were sacrificed on GD 21 and gross lesions and the weight of thyroid gland were recorded. Pregnancy status and the numbers of corpora lutea, implantations, resorptions, and live and dead fetuses were noted. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal abnormalities.

There were no mortalities associated with TMC278. There were no relevant clinical signs. Reduced body weight gain (maximally 7%) and food consumption (maximally 4%) were noted in dams given 120 and 400 mg/kg/day. Weight of thyroid gland showed a dose-related increase (maximally 24%), which was statistically significant in the animals dosed with 120 and 400 mg/kg/day. There were no effects on gross pathology or pregnancy parameters; numbers of corpora lutea; implantations; early and late resorptions; live or dead fetuses; or on



the pre- or postimplantation losses. There were also no effects on fetal body weight or sex ratio. Visceral examinations showed a slight increase in dilated renal pelvis in 5 out of 149 and 7 out of 149 fetuses from the groups treated with 120 and 400 mg/kg/day, respectively. The dilated renal pelvis is considered a minor variation. The occurrences of the 2 malformations in 2 different litters of dams treated with 400 mg/kg/day noted upon external examinations were isolated and dissimilar in nature, and bore no relationship with treatment. In view of the isolated nature and the possible occurrence within a normal control population, these were not considered to be test article related.

The maternal and embryo fetal NOAEL was 40 mg/kg/day based on the changes on the body weight, food consumption, and the increase of dilated renal pelvis seen at higher doses. Systemic exposure expressed as Cmax and AUC values are given in Table 25 below.

Table 25. Exposure to TMC278 (Cmax and AUC) in an Oral Embryo-Fetal Developmental Study with TMC278 Base in Rats

Cmax (μg/Ml) AUC0 24h (μg.h/Ml) Dose

(mg/kg/day) Sampling Day F (n = 6)

GD 6 4.9 33 40

GD 16 5.6 37

GD 6 6.0 65 120

GD 16 7.2 63

GD 6 14 182 400

GD 16 13 152

F: females

2.6.6.6.2.6. Rilpivirine: Embryo-Fetal Development Studies in Rabbits (with TMC278 Base)

The doses for the main rabbit embryo-fetal development study were selected on the basis of the results of 2 pilot studies. In the first study (Tabulated Summary 2.6.7.11.B, TMC278-NC128), 75 and 150 mg of TMC278 base/kg/day from GDs 6 to 19 incurred complete resorption of litters. In the second study (Tabulated Summary 2.6.7.11.B, TMC278-NC129), animals were dosed with 0 (0.5% aqueous HPMC), 5, 20, or 60 mg of TMC278 base/kg/day from GDs 6 to 19. The dose of 60 mg/kg/day resulted in reduced fetal survival and postimplantation loss without external fetal abnormalities.

In the main study, TMC278 base suspended in aqueous 0.5% (m/v) HPMC was administered once daily, by oral gavage, from GDs 6 to 19 to groups of 20 pregnant New Zealand white rabbits at 0 (vehicle), 5, 10, or 20 mg/kg/day in a dose volume of 5 mL/kg (Tabulated Summary 2.6.7.13.D, TMC278-NC130). Blood samples for toxicokinetic analysis were collected from 3 animals per group on GDs 6 and 19 at 7 time points during the 24 hours after dosing. Clinical signs, body weight, and food consumption performance were



recorded. The females were killed on GD 28 and a necropsy was performed. The females were examined for gross lesions, pregnancy status, the numbers of corpora lutea of pregnancy, implantations, resorptions, and live and dead fetuses. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal abnormalities.

There were no mortalities. There were no relevant clinical signs, no effects on body weight or food consumption, and no gross lesions at necropsy. Pregnancy parameters as assessed by the number of corpora lutea of pregnancy, implantations, resorptions, live and dead fetuses, or pre- or postimplantation loss were unaffected. Moreover, there were no effects on mean fetal body weight or on the sex ratio. There were no relevant changes at external, visceral, or skeletal fetal examination, except for a slight increase in numbers of fetuses exhibiting branches of the left subclavian artery originating from the aorta and hypoplastic interparietal bone which reached statistical significance in the group receiving 20 mg/kg/day. Since no clear dose-related increase in incidence of hypoplastic interparietal bones was observed, the latter finding was considered of minor toxicological importance. The aortic and pulmonary trunks and their respective branching are particularly ‘plastic’ in the fetus and are susceptible to modification, especially in the rabbit. As these changes are commonly seen in rabbits, they are considered to have little or no biological significance.

The maternal NOAEL was at least 20 mg/kg/day and the fetal NOAEL was 10 mg/kg/day. Systemic exposure expressed as Cmax and AUC values are given in Table 26 below (Tabulated Summary 2.6.7.13.D, TMC278-NC130).

Table 26. Exposure to TMC278 (Cmax and AUC) in an Oral Embryo-Fetal Development Study with TMC278 Base in Rabbits


(mg/kg/day) Sampling Day F (n = 3)

GD 6 6.4 95 5

GD 19 6.7 105

GD 6 9.7 162 10

GD 19 10 170

GD 6 13 219 20

GD 19 15 232

F: females



2.6.6.6.2.7. Tenofovir DF: Dose Range Pilot Study in Rats

Eight pregnant Crl:CD BR VAF/PIus (Sprague-Dawley) rats in each of 4 dosage groups were dosed (via gavage) once daily, on GDs 7 through 17 with 30, 100, 300, and 1000 mg/kg/day of TDF, respectively (Tabulated Summary 2.6.7.11.C, 97-TOX-4331-003). An additional 8 pregnant rats were dosed with suspension vehicle. Clinical observations and body weights were taken daily and feed consumption was recorded on GDs 0, 7, 10, 12, 15, 18, and 20. All surviving rats were cesarean-sectioned on GD 20 and fetal viability, body weight, sex, and gross external observations were evaluated.

One high-dosage group rat was found dead on GD 12. This rat had excess salivation, rales, ungroomed coat, localized alopecia, urine-stained abdominal fur, and soft or liquid feces prior to death. Gross necropsy revealed agonal changes of enlarged adrenal glands and dark red intestine. All 14 conceptuses in this rat appeared normal for their developmental age at the time of maternal death. All other rats survived to cesarean-sectioning on GD 20. Decreased maternal body weight and/or body weight gain, decreased food consumption, and excess salivation were observed in the 300 and 1000 mg/kg/day dosage groups. The 1000 mg/kg/day dosage group rats also had urine-stained abdominal fur, rales, ungroomed coat, red perivaginal substance, and emaciated appearance. Cesarean-sectioning revealed decreased fetal weight at 1000 mg/kg/day. All other developmental parameters evaluated were within the historical range of the testing facility; there were no fetal gross observations related to test article administration.



2.6.6.6.2.8. Tenofovir DF: Oral (Gavage) Developmental Toxicity Study of GS-4331-05 in Rats

Twenty-five Crl:CD®BR VAF/PIus® (Sprague-Dawley) presumed pregnant female rats were assigned to each of 4 groups (groups I through IV) (Tabulated Summary 2.6.7.13.E, 97-TOX-4331-004). The test article, TDF (GS-4331-05), or the suspension vehicle was administered via oral gavage once daily to female rats on GDs 7 through 17. Dosages of 0 (Vehicle), 50, 150, and 450 mg/kg/day of the test article were administered at a dosage volume of 10 mL/kg, adjusted daily on the basis of the individual body weights recorded before dosing. The rats were intubated once daily at approximately the same time each day. Dosages were selected based on results from the dosage-range developmental toxicity study of TDF in rats (97-TOX-4331-003), which showed mortality in 1 dam (1000 mg/kg/day); decreased maternal body weight and/or body weight gain (300 and 1000 mg/kg/day); decreased fetal body weights (1000 mg/kg/day); and maternal clinical signs of toxicity (1000 mg/kg/day). The female rats were observed for viability at least twice each day of the study and for general appearance on GD 0. The rats were also examined for clinical observations of effects of the test article, abortions, premature deliveries, and deaths before and approximately 30 minutes after dosage administration. These observations were also conducted once daily during the postdosage period. Body weights were recorded on GD 0 and daily during the dosage and postdosage periods. Feed consumption values were recorded on GDs 0, 7, 10, 12, 15, 18, and 20. All rats were sacrificed by carbon dioxide asphyxiation on GD 20, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. The number of corpora lutea in each ovary was recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantations, live and dead fetuses, and early and late resorptions. Each fetus was weighed and examined for sex and gross external alterations. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations. The remaining fetuses in each litter were eviscerated and examined for skeletal alterations.

All rats survived until scheduled sacrifice. A statistically significant increase in the incidence of localized alopecia on the underside occurred in the 450-mg/kg/day dosage group. Although this finding is considered to be of minimal biological importance, it was considered an effect of the high dosage of the test article because: (1) the incidence was statistically significant; (2) the observation occurred only in high dosage group rats; and (3) the observation occurred in one 1000 mg/kg/day dosage group rat that died in the range-finding study. All other adverse clinical and necropsy observations were considered unrelated to dosages of the test article as high as 450 mg/kg/day. The 450 mg/kg/day dosage of the test article resulted in a transient, statistically significant weight loss in the dams on GDs 7 to 10. Body weight gains did not significantly differ among the 4 dosage groups throughout the remainder of the dosage period. Reflecting the initial weight loss in the high dosage group, body weight gain was significantly reduced for the entire dosage period (calculated as GDs 7 to 18), the entire interval after the first dosage (GDs 7 to 20), and the entire gestation period (GDs 0 to 20). Maternal body weights were significantly reduced on GDs 9 through 20. The 450 mg/kg/day dosage of the test article significantly reduced absolute (g/day) and relative (g/kg/day) maternal feed consumption values on GDs 7 to 12; the absolute feed consumption value was also significantly reduced on GDs 15 to 18. This effect of the test article was most severe on



GDs 7 to 10 and resulted in significant reductions in absolute and relative feed consumption values for the entire dosage period. After completion of the dosage period, absolute and/or relative feed consumption values were significantly increased in all groups administered the test article; rebound effects commonly observed in these types of studies after completion of the dosage period. Despite these increases in feed consumption values after completion of the dosage period, absolute and relative feed consumption values were significantly reduced in the 450-mg/kg/day dosage group for the entire period after the first dosage was administered (GDs 7 to 20), and the absolute feed consumption value was significantly reduced for the entire gestation period (GDs 0 to 20).

Cesarean-sectioning and litter values were unaffected by dosages of the test article as high as 450 mg/kg/day. The litter averages for corpora lutea, implantations, live fetuses, total and early resorptions, percent live male fetuses (sex ratio), fetal body weights, and percent resorbed conceptuses and the numbers of dams with any resorptions or with viable fetuses were comparable among the 4 dosage groups and did not significantly differ. All placentae appeared normal. All gross external, soft tissue, and skeletal alterations (malformations and variations) were considered unrelated to administration of dosages of the test article as high as 450 mg/kg/day. Fetal ossification site averages were unaffected by dosages of the test article as high as 450 mg/kg/day. The litter averages per site per fetuses were comparable among the 4 dosage groups and did not significantly differ.

Based on the results of this study, the maternal NOEL for TDF is 150 mg/kg/day. Maternal toxicity was evident in the 450 mg/kg/day dosage group as adverse clinical observations (localized alopecia), transient body weight loss, and reductions in feed consumption. The fetal developmental NOEL is 450 mg/kg/day (no effects were observed at the highest dosage tested).

2.6.6.6.2.9. Tenofovir DF: Oral (Stomach Tube) Dose-Range Finding Developmental Toxicity Study of GS-4331-05 (bis-POC PMPA) in Rabbits

Five pregnant New Zealand White [Hra:(NZW)SPF] rabbits in each of 3 dosage groups were dosed (via stomach tube) once daily on DGs 6 to 18 with 30, 100, and 300 mg/kg/day of TDF, respectively (Tabulated Summary 2.6.7.11.C, 97-TOX-4331-006). An additional 5 pregnant rabbits were dosed with suspension vehicle. Clinical observations, body weights, and feed consumption values were recorded daily. All surviving rabbits were cesarean-sectioned on GD 29. The uterus was opened and examined for placental abnormalities, the number and placement of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. Fetal viability, body weight, sex, and gross external observations were evaluated. Maternal kidneys were weighed and retained.

Clinical observations considered to be treatment-related were limited to red substance in the cage pan and soft or liquid feces observed in 1 doe from the 300-mg/kg/day dosage group which aborted on GD 27. There were no treatment-related necropsy observations. Maternal body weight loss occurred at the 100-mg/kg/day dosage level on GDs 9 to 12 and at the 300-mg/kg/day dosage level for the entire dosage period (GDs 6 to 19). Body weight gains were reduced for the 300-mg/kg/day dosage group for the entire gestation period (GDs 0 to



29) and the entire gestation period after the initiation of dosage (GDs 6 to 29). Absolute and relative feed consumption values for the 100- and 300-mg/kg/day dosage groups were reduced during the dosage period and slightly reduced for the entire gestation period after the initiation of dosage (GDs 6 to 29). Maternal kidney weights were slightly decreased in the 300-mg/kg/day dosage group (87.8% of the control group value). No cesarean-sectioning or litter parameters were affected by administration of TDF (bis-POC PMPA) to does at dosages as high as 300 mg/kg/day. All fetuses appeared normal at fetal gross evaluation.

2.6.6.6.2.10. Tenofovir DF: Oral (Stomach Tube) Developmental Toxicity Study of GS-4331-05 (bis-POC PMPA) in Rabbits

One-hundred and thirty artificially inseminated New Zealand White (Hra:[NZW]SPF) rabbits were randomly assigned to 4 dosage groups (Groups I through IV), including 35 rabbits per Groups I, II, and IV, and 25 per group IIIb (b: an additional 10 artificially inseminated female rabbits were assigned to the 0- [vehicle], 30-, 100-, and 300-mg/kg/day dosage groups due to a low pregnancy rate in the main study [7, 6, and 7 rabbits were not pregnant in the 3 respective dosage groups]; Tabulated Summary 2.6.7.13.F, 98-TOX-4331-005). Four rabbits per group were assigned to the satellite portion of the study for blood collection. The test article, TDF or the vehicle (suspension vehicle), was administered orally (via stomach tube) to these rabbits on GDs 6 through 18 at dosages of 0 (vehicle), 30, 100, or 300 mg/kg/day. The dosage volume was 10 mL/kg, adjusted daily for body weight changes and given at approximately the same time each day. All rabbits were observed for viability at least twice each day of the study and for general appearance weekly during acclimation and on GD 0. Additional examinations for clinical observations of effects of the test article, abortions, premature deliveries, and deaths were made daily before and approximately 60 10 minutes after administration during the dosage period and once daily during the postdosage period. Body weights were recorded on GD 0 and daily during the dosage and postdosage periods. Feed consumption values were recorded daily throughout the study. Approximately 1 mL of blood was collected from the satellite rabbits on GD 18 at 0 (predosage), 0.5, 1, 2, 4, 8, 12, and 24 hours postdosage for analysis of pharmacokinetic parameters. The rabbits assigned to the satellite portion of the study were sacrificed on GD 19 following the 24-hour blood collection, necropsied as described for main study rabbits, and examined for pregnancy status. Uterine contents were recorded and kidneys were retained. All fetuses were discarded following examination. All surviving rabbits assigned to the main portion of the study were sacrificed on GD 29. The rabbits were cesarean-sectioned and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. Gross lesions were retained and kidneys were excised, weighed (paired weight), and retained. The number of corpora lutea in each ovary was recorded. The uterus was excised and examined for pregnancy, number and distribution of implantations, early and late resorptions, and live and dead fetuses. Each fetus was weighed and examined for gross external alterations. All fetuses were examined internally to identify sex and visceral alterations. All fetuses were also examined for skeletal alterations.



One doe in the 300 mg/kg/day dosage group aborted and was sacrificed on GD 20. This abortion was considered test article-related because it occurred in the highest dosage group. All other does survived until scheduled sacrifice. The number of does with scant feces in the 300-mg/kg/day dosage group was significantly increased. A red substance in the cage pan was observed in 2 rabbits from the 300-mg/kg/day dosage group on GDs 19 and 20. No necropsy observations were considered test article-related. Maternal body weight gains were significantly reduced in the 300-mg/kg/day dosage group for the entire dosage period (calculated as GDs 6 to 19). During the postdosage period (GDs 19 to 29), body weight gains were significantly increased in the 300-mg/kg/day dosage group; a rebound phenomenon that commonly occurs in these types of studies. Significant body weight loss or reduced body weight gains occurred in the 300-mg/kg/day dosage group on GDs 7 to 8, 9 to 12, and 15 to 19. Maternal body weights were also significantly reduced on GD 19. Maternal absolute (g/day) and relative (g/kg/day) feed consumption values were significantly reduced in the 300-mg/kg/day dosage group for the entire dosage period (calculated as GDs 6 to 19); for the entire gestation period after the initiation of dosage administration (GDs 6 to 29); and for each tabulated interval during the dosage period. No cesarean-sectioning or litter parameters were affected by dosages of the test article as high as 300 mg/kg/day. There were no dosage-dependent or biologically important differences in the litter or fetal incidences of any gross external, soft tissue, or skeletal alterations.

Based on these data, the maternal NOEL for the TDF is 100 mg/kg/day (the 300 mg/kg/day dosage caused adverse clinical observations and reductions in maternal body weight gain and feed consumption). The developmental NOEL is greater than 300 mg/kg/day (no effects occurred at the highest dosage tested).


Concentrations of TFV were determined in plasma samples obtained during the course of a developmental toxicology study in rabbits (Tabulated Summary 2.6.5.7.G, 98-TOX-4331-005-PK). Pregnant rabbits (n = 4 per group) received TDF (at 30, 100, or 300 mg/kg/day) by oral gavage on GDs 6 to 18. Blood samples were collected on GD 18, immediately prior to TDF administration and at 0.5, 1, 2, 4, 8, 12, and 24 hours postdose. Blood was immediately processed for plasma and maintained frozen ( 20 5 °C) until analysis. On GD 19, the rabbits were sacrificed and their pregnancy status examined. Only data from animals that were determined to be pregnant were used in the study. Following repeated oral administration of TDF on GDs 6 through 18, concentrations of TFV in plasma on GD 18 reached mean peak values of 4.92 1.34, 20.65 4.09, and 47.62 9.51 g/mL at the 30, 100, and 300 mg/kg/day dose levels, respectively. The Tmax in plasma was similar for each dose level; 0.75 hour at dose levels of 30 and 300 mg/kg/day, and 0.5 hour at the dose level of 100 mg/kg/day. There was a biphasic decline in plasma concentrations with time, with an apparent terminal half-life of 8.61 1.64, 10.34 3.08, and 9.67 3.23 at dose levels of 30, 100, and 300 mg/kg/day, respectively. Tenofovir Cmax and AUCtau appeared to increase in a dose proportional manner following repeated dose administration of TDF, indicating linear pharmacokinetics following multiple daily administrations to pregnant rabbits.



2.6.6.6.3. Prenatal and Postnatal Development Including Maternal Function

2.6.6.6.3.1. Emtricitabine: Pre- and Postnatal Development Study in CD-1® Mice Given TP-0006 by Gavage

Groups of 25 pregnant female CD-1 mice were given FTC by oral gavage at 0, 250, 500, and 1000 mg/kg/day (5 mL/kg/dose; 10 mL/kg/day) (Tabulated Summary 2.6.7.14.A, TOX039). Doses were administered to F0 dams BID, with approximately 6 hours between equally divided doses, from GD 6 through postnatal day (PND) 20. The F0 dams raised their pups through weaning at PND 21, at which time the F1 weanlings were allowed to mature to at least 10 weeks of age. During maturation, attainment of sexual development (preputial separation and vaginal patency) and neurodevelopmental milestones (motor skills, learning, and memory) were monitored. Immediately prior to mating, estrous cyclicity of F1 females was evaluated. F1 animals were then cohabited within dose groups (sibling matings avoided) and the resulting F2 litters were evaluated and the pups sacrificed.

There was no treatment-related toxicity observed in the F0 dams during gestation or lactation, including mortality, body weight changes, or reduced feed consumed. The F1 litters were normal at all doses tested with respect to number of pups, pup weights, and sex ratios. Maturation of F1 pups through weaning proceeded normally. Attainment of neurodevelopmental and sexual development milestones were all normal. Body weights of F1 animals during the postweaning maturation phase were normal across all groups. F1 dams in the 1000-mg/kg/day group had slightly longer estrous cycles (0.64 days) than controls. Survival, body weights, and sex ratios of F2 litters were normal across all groups. Necropsy of F1 animals yielded no treatment-related findings. In summary, the NOAEL for reproductive toxicity in the mouse under the conditions of this study was found to be

1000 mg/kg/day.

Exposure following oral administration of FTC to pregnant mouse dams and the exposure of murine fetuses to FTC was examined in a separate study (Module 2.6.4, Section 2.6.4.4.4.1;Tabulated Summary 2.6.5.7.A, TOX103). On GD 15 following approximately 10 days administration of 500 mg/kg of FTC BID, pregnant mice and their viable fetuses had measurable concentrations of FTC at 1 hour following administration of the first 500-mg/kg dose. The mean ( SD) plasma concentration in the pregnant mice was 137.1 28.0 g/mL. The mean concentration of FTC in pooled fetal homogenate was 55.7 10.4 g/g. The mean fetal/maternal concentration ratio was 0.41 0.04.

2.6.6.6.3.2. Rilpivirine: Dose Range Finding Peri- and Postnatal Development Study Including Juvenile Exposure in Rats with TMC278

The objectives of this pilot study were to investigate the tolerability of doses applied in the general and reproduction toxicity studies on pre- and postnatal development parameters (Tabulated Summary 2.6.7.14.B, TMC278-NC168). Moreover, the effects of direct administration of TMC278 to pups for 2 weeks were evaluated in terms of exposure and postnatal development.



TMC278 suspended in 0.5% m/v aqueous HPMC was administered once daily by oral gavage to groups of 6 time-mated female Sprague Dawley rats from GD 6 to LD 7 (day of delivery is LD 0) at 0 (vehicle, 2 groups), 40, 120, and 400 mg/kg/day in a dose volume of 10 mL/kg. Regular observations were made for maternal clinical signs, body weight, and food consumption. The females were allowed to litter. Observations on nesting and nursing behavior were made. On LD 7, 8 male and 8 female pups of each group were selected for oral dosing by gavage with the same dose and at the same dose volume as their mothers. The selected pups from the second vehicle group were dosed with 400 mg/kg/day. Blood samples were taken from 12 nonselected male and 12 nonselected female pups of each group on LD 7. These pups were killed after the sampling. The selected pups were dosed from LD 12 up to and including LD 25. They were observed regularly for clinical signs, morbidity, and mortality, and their body weight was recorded. Blood samples for toxicokinetics were taken on LD 25. Necropsy was performed after the last sampling.

There were no mortalities among the dams. Moreover, the dams showed no abnormal clinical signs or effects on body weight or food consumption. There were no changes on pregnancy or litter parameters. Two pups from the group treated with 400 mg/kg/day were killed for humane reasons. There were no effects on the pups treated directly with TMC278 during the dosing period or at necropsy. Systemic exposure for pups expressed as Cmax and AUC values are given in Table 27 below.

Table 27. Exposure to TMC278 (Cmax and AUC) in Rat Pups via Lactation (LD7) and after 2 Weeks of Oral Dosing with TMC278


(mg/kg/day) Sampling Day M F M F

LD7 a 0.05 0.07 0.62 0.74 40

LD25 b 2.6 5.8 12 18

LD7 0.08 0.08 0.94 0.91 120

LD25 3.7 3.6 34 28

LD7 0.16 0.14 1.9 1.8 400

LD25 9.1 7.3 50 53

400c LD25 6.4 4.9 43 39

M: males, F: females, LD: lactation day, a: pups from dams dosed as indicated exposed in utero and via lactation (n = 12/sex), b: pups from dams as indicated exposed in utero and via lactation and dosed by gavage from LD12 to LD25 (n = 8/sex), c: pups from control dams dosed by gavage from LD12 – LD25 (n = 8/sex)



2.6.6.6.3.3. Rilpivirine: Peri- and Postnatal Developmental Toxicity Study in Rats with TMC278

TMC278 suspended in 0.5% (m/v) aqueous HPMC was administered once daily by oral gavage to groups of 25 time-mated female Sprague Dawley rats from GD6 to LD 20 at 0 (vehicle), 40, 120, or 400 mg/kg/day in a dose volume of 10 mL/kg (Tabulated Summary 2.6.7.14.C, TMC278-NC131). Regular observations were made for maternal clinical signs, morbidity, and mortality. Weekly body weight and food consumption were determined. The females were allowed to litter. Litter size and sex ratio were determined. Pups weight was determined regularly. Developmental landmarks, including number of ears open, static righting reflex, eyes open, startle response, and pupillary light reflex were recorded on LDs 3, 5, 15, and 21. At necropsy of the dams after weaning of their litters, the number of implantation scars in uterus was determined. From the F1 generation, 20 males and 20 females per group were kept and their growth, postweaning development, behavior, and reproductive performance were assessed.

There were no mortalities among the dams. The dams showed no relevant clinical signs and no effects on body weight, food consumption, gestation, or parturition. There were no effects on the numbers of pups born, the sex ratio, pup survival, or physical condition. There were no effects on pre- or postweaning body weight and gain, or development and behavior parameters. Sexual development, fertility, mating performance, and pregnancy parameters of the F1 generation were unaffected by treatment of the F0 generation. There were no effects on postmortem parameters at necropsy of the F0 and F1 generation.

The NOAEL for both the F0 and F1 generation was at least 400 mg/kg/day.

2.6.6.6.3.4. Tenofovir DF: Oral (Gavage) Developmental and Perinatal/Postnatal Reproduction Toxicity Study Of GS-4331-05 (Bis-Poc PMPA) in Rats, Including a Postnatal Behavioral/Functional Evaluation

One hundred twenty-five presumed pregnant Crl:CD®(SD)IGS BR VAF/Plus® rats were randomly assigned to 5 dosage groups (Groups I through V), 25 rats per group (F0 generation rats; Tabulated Summary 2.6.7.14.D, R990202). An additional 12 rats were assigned to each of Groups II through V for satellite toxicokinetic evaluations. Suspensions of the test article, TDF (GS-4331-05), or the suspension vehicle were administered orally once daily to these naturally-bred female rats from GD 7 through LD 20, or GD 24 (rats that did not deliver a litter), at dosages of 0 (vehicle), 50, 150, 450, and 600 mg/kg/day. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately before administration. Rats were observed for viability at least twice each day of the study. Observations for clinical signs of effects of the test article, abortions, premature deliveries, and deaths were made daily before dosage and approximately 60 10 minutes after dosage administration. Body weights were recorded GD 0, daily during the dosage period, and at sacrifice. Feed consumption values were recorded on GDs 0, 7, 10, 12, 15, 18, 20, and 25 (if necessary) and LDs 1, 4, 7, 10, and 14. Rats were evaluated for adverse clinical signs observed during parturition, duration of gestation, litter sizes, and pup viability at birth. Maternal behavior was evaluated on LDs 1, 4, 7, 14, and 21. On GD 7 and LD 20, blood



samples were collected predosage, at 15 and 40 minutes, and at 1, 2, 4, 6, 8, 12, and 24 hours postdosage. Milk samples were collected from rats assigned to the satellite portion of the study and from 1 vehicle control group rat approximately 1 hour postdosage on LD 11. After completion of the 21-day postpartum period, female rats were sacrificed and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. Kidneys were excised, weighed (paired weight), and retained for possible histologic examination. The number and distribution of implantation sites was recorded. Gross lesions were retained for possible future evaluation.

Each F1 generation litter was evaluated for viability at least twice each day. The pups in each litter were counted once each day. Clinical observations were recorded once daily during the preweaning period. Pup body weights were recorded on LDs 1, 4, 7, 14, and 21. On LD 21, all pups not selected for continued evaluation were sacrificed and examined for gross lesions. The following tissues were excised and retained for possible histologic evaluation: kidneys (paired weight recorded); sternum (with costochondral junction); and femur. Necropsy of the pups included a single cut at the suture of the frontal and parietal bones of the skull, and the cross-sectioned brain was examined for hydrocephaly. Tissues with gross lesions were retained for possible future evaluation.

All F1 generation male and female rats were observed for viability at least twice each day of the study. These rats were also examined for clinical observations and general appearance once weekly during the postweaning period. Body weights for male rats were recorded weekly during the postweaning period and at sacrifice. Body weights for female rats were recorded weekly during the postweaning period and on GDs 0, 7, 10, 14, 17, and 20. Feed consumption values for male rats were recorded weekly, except during cohabitation. Feed consumption values for female rats were recorded weekly during the postweaning period, except during cohabitation and on GDs 0, 7, 10, 14, 17, and 20.

Female rats were evaluated for the age of vaginal patency, beginning on Day 28 postpartum. Male rats were evaluated for the age of preputial separation, beginning on Day 39 postpartum. Beginning at 24 1 day postpartum, 1 male rat and 1 female rat from each litter, where possible, were evaluated in a passive avoidance test for learning, short-term retention, and long-term retention. Beginning at approximately 70-days postpartum, 1 male rat and 1 female rat from each litter were evaluated in a water-filled M-maze for overt coordination, swimming ability, learning, and memory. At approximately 90 days of age, the F1 generation rats were assigned to cohabitation.

Male rats were sacrificed after completion of the 21-day cohabitation period. A gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. Testes and epididymides of male rats were excised and paired organ weights were recorded. The epididimides were retained in neutral buffered 10% formalin. The testes were fixed in Bouin’s solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. Female rats were sacrificed on GD 20, cesarean-sectioned, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. The rats were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late



resorptions. Each fetus was weighed and examined for sex and gross external alterations. All fetuses were retained in alcohol.

No deaths related to the test article, TDF (bis-POC PMPA), occurred in the F0 generation rats. One rat in the 150 mg/kg/day dosage group was found dead. One rat in each of the 0 (vehicle), 450, and 600 mg/kg/day dosage groups were moribund sacrificed. All of these deaths were considered related to intubation errors, with the exception of the vehicle control group rat, which appeared to have dystocia. All other rats survived until scheduled sacrifice. Clinical observations of red perioral substance and excess salivation occurred in an increased or statistically significantly (p 0.05 to p 0.01) increased number of rats in the 150, 450, and 600 mg/kg/day dosage groups during the gestation period. Excess salivation and perioral substance were not considered related to treatment and probably did not reflect systemic toxicity of the test article. Test article-related clinical observations during gestation and urine-stained abdominal fur was observed in statistically significantly (p 0.05 to p 0.01) increased numbers of rats in the 450 and 600 mg/kg/day dosage groups, and ungroomed coat occurred in a statistically significantly (p 0.01) increased number of rats in the 600 mg/kg/day dosage group. Two rats in each of the 450 and 600 mg/kg/day dosage groups had soft or liquid feces during gestation. Observations of urine-stained abdominal fur, red perioral substance, and ungroomed coat continued in statistically significantly increased numbers of rats in the 450 and 600 mg/kg/day dosage groups during the lactation period. Additionally, dehydration and emaciation occurred in increased or statistically significantly increased numbers of rats in the 450 and 600 mg/kg/day dosage groups, and cold to touch occurred in a significant number of 600 mg/kg/day dosage group rats during lactation. Soft or liquid feces occurred in 4 rats in each of the 450 and 600 mg/kg/day dosage groups during lactation. All other clinical observations were considered unrelated to the test article.

All necropsy observations were considered unrelated to administration of the test article. Terminal body weights, paired kidney weights, and the ratios of the kidney weights to the terminal body weights were comparable among the 5 dosage groups and did not statistically significantly differ. Body weight gains for the entire gestation dosage period (GDs 7 to 20) and the entire gestation period (GDs 0 to 20) were statistically significantly reduced in the 150, 450, and 600 mg/kg/day dosage groups. Gestation body weights were statistically significantly reduced in the 150 mg/kg/day dosage group on GDs 19 and 20; in the 450 mg/kg/day dosage group on GDs 16 through 20; and in the 600 mg/kg/day dosage group on GDs 10 through 20. During the lactation period, maternal body weight gains tended to be increased over or comparable to the control group values in the 150, 450, and 600 mg/kg/day dosage groups and were statistically significantly increased in the 450 and 600 mg/kg/day dosage groups for the entire lactation period (LDs 1 to 21). Reflecting the reductions in body weight gain that occurred during the gestation period, lactation body weights continued to be statistically significantly reduced in the 450 mg/kg/day dosage group on LDs 1 through 6 and 8 and in the 600 mg/kg/day dosage group on LDs 1 through 5 and 7 through 9. Absolute (g/day) and relative (g/kg/day) feed consumption values during gestation were statistically significantly reduced in the 450 and 600 mg/kg/day dosage groups for the entire gestation dosage period (GDs 7 to 20) and the entire gestation period (GDs 0 to 20). During lactation, significant reductions in absolute and relative feed consumption values occurred in the 450 (absolute only) and 600 mg/kg/day dosage groups for the entire tabulated lactation period



(LDs 1 to 14). Peripartum/postpartum pup mortality and stillbirths were increased in the 450 and 600 mg/kg/day dosage groups, and there was a concomitant significant reduction in the percentage of liveborn pups in the 600 mg/kg/day dosage group. The percentages of pups found dead or presumed cannibalized were increased or statistically significantly increased on LDs 1 and 2 to 4 in the 450 and 600 mg/kg/day dosage groups; and a significant number of dams had no surviving pups on LDs 2, 3, or 4 in these 2 dosage groups (5 and 10, respectively). Additionally, one dam in the 600 mg/kg/day dosage group had no surviving pups on LD 5. As a result of these pup deaths, the Viability Index was reduced in the 450 mg/kg/day dosage group and statistically significantly reduced in the 600 mg/kg/day dosage group. A significant number of pups were found dead or presumed cannibalized on LDs 8 to 14 in the 450 mg/kg/day dosage group and there was a corresponding slight reduction in the Lactation Index. Pup weights were statistically significantly reduced on LDs 1, 4, 7, 14, and 21 in the 450 and 600 mg/kg/day dosage groups. Increased or statistically significant increased numbers (compared to the control group values) of F1 generation litters had pups that were cold to touch, pale, and had umbilical hernias in the 450 and 600 mg/kg/day dosage groups. A statistically significant increased number of litters had dams and pups that were not nesting in the 600 mg/kg/day dosage group. An increased number of pups that were found dead and necropsied in the 450 and 600 mg/kg/day dosage groups had no milk in the stomach. Weights of the kidneys were statistically significantly reduced in the 450 and 600 mg/kg/day dosage groups, reflecting the statistically significantly reduced average body weights in these groups on LD 21. Ratios of the kidney weights to the terminal body weights were comparable among the 5 dosage groups. All other natural delivery and litter observations were unaffected by dosages of the test article as high as 600 mg/kg/day.

Of the F1 generation rats, 1 male rat in the 450-mg/kg/day group and 2 male rats in the 600 mg/kg/day dosage group, respectively, were found dead. One male rat in the 600 mg/kg/day dosage group was moribund sacrificed. Two female rats in each of the 450 and 600 mg/kg/day dosage groups were found dead. One female rat in the 450 mg/kg/day dosage group was moribund sacrificed. The F1 male death in the 150 mg/kg/day dosage group was not considered related to maternal test-article exposure. The deaths in the 450 and 600 mg/kg/day dosage groups were considered related to the test article because: (1) they occurred in F1 generation rats at maternally toxic dosages of 450 and 600 mg/kg/day; and (2) the incidences were dosage-dependent. These rats are discussed below. All other F1 generation rats survived until scheduled sacrifice. There were no adverse clinical observations in the F1 generation male and female rats that were considered to be test article related. Necropsy observations of empty stomach, small and large intestines distended with gas, dark red area on the glandular mucosal surface of the stomach, and tan spleen were considered to be associated with dead or moribund sacrificed F1 generation male and female rats. Dosage-dependent reduced to statistically significantly reduced terminal body weights of the F1 generation male rats were observed at all dosage levels. Paired testes weights were reduced and statistically significantly reduced in the 450 and 600 mg/kg/day dosage groups, and the paired epididymides weight was statistically significantly reduced for the 600 mg/kg/day dosage group. Paired testes weights were also reduced, although not statistically significantly reduced in the 50 and 150 mg/kg/day dosage groups. The result was statistically significantly increased epididymides to body weight ratio for the 150, 450, and 600 mg/kg/day dosage groups. The significant reduction in the weight of the epididymides in



the 600 mg/kg/day dosage group was not considered biologically important because the mating and fertility of the male rats in this dosage group were unaffected. Reflecting the reductions in pup body weights during the preweaning period, body weights and body weight gain of the F1 generation rats in all treated groups were reduced or statistically significantly reduced during the postweaning period (Days 1 and 71 postweaning). Gestation body weights for the F1 generation female rats were reduced to statistically significantly reduced in all treated groups. Gestation body weight gains were comparable for the 5 groups of the F1 generation female rats. Absolute feed consumption values during the postweaning period for the F1 generation males were statistically significantly reduced in all treated groups. Absolute feed consumption values during the postweaning period for the F1 generation females were statistically significantly reduced over the postweaning period Days 8 to 15 by maternal dosages of 600 mg/kg/day of the test article. Absolute feed consumption values during the postweaning period for the F1 generation females were comparable for all 5 groups, with the exception noted above. Relative feed consumption values during the postweaning period for the F1 generation females were increased to statistically significantly increased for the 50 mg/kg/day dosage group and above from Days 1 to 71 postweaning. These changes were not considered biologically significant for the 50 mg/kg/day dosage group. The average day on which preputial separation was evident in male rats was statistically significantly increased and was dosage-dependent for F1 generation male rats whose dams were administered dosages of the test article of 450 and 600 mg/kg/day. The time to reach vaginal patency in female rats was statistically significantly prolonged (450 mg/kg/day group) or prolonged (600 mg/kg/day group) for F1 generation females whose dams were administered 450 or 600 mg/kg/day, respectively.

In the passive avoidance paradigm, F1 male and female rats the 450 mg/kg/day dosage group had first trial latencies that were statistically significant increased. This effect was not dosage-dependent, probably because of the few animals tested in the 600 mg/kg/day dosage group. No other biologically important differences occurred for short- or long-term memory or learning in this paradigm. There were no biologically important differences in the values for learning, short-term retention, long-term retention, or response inhibition in the F1 generation male or female rats, as evaluated by performance in a watermaze swim task. The biologically important differences in the values for learning, short-term retention, long-term retention, or response inhibition in the F1 generation males and females from the 450 and 600 mg/kg/day groups were considered improved performance for the passive avoidance paradigm compared to the vehicle group. Mating performance of the F1 generation male and female rats was unaffected by administration of the test article to the F0 generation dams at dosages as high as 600 mg/kg/day. Cesarean-sectioning and litter parameters in the F1 generation female rats were unaffected by administration of dosages of the test article as high as 600 mg/kg/day to the F0 generation dams.



On the basis of these data, the maternal NOEL of TDF is 50 mg/kg/day. The 150, 450, and 600 mg/kg/day dosages caused adverse clinical observations, reductions in body weight gain during gestation, and increases in body weight gain during lactation; and the 450 and 600 mg/kg/day dosages reduced gestation and lactation feed consumption values. The developmental NOEL is 150 mg/kg/day. The 450 and 600 mg/kg/day dosages resulted in increased peri/postpartum pup mortality, reduced pup survival, and reduced pup body weights. The NOEL for general toxicity of TDF in the F1 generation is 50 mg/kg/day (mortality occurred or body weights and/or feed consumption were reduced in the 150, 450, and 600 mg/kg/day dosage groups). The F1 generation male and female NOEL for behavioral, reproductive, and developmental toxicity of TDF is 150 mg/kg/day (effects on sexual maturation were observed at dosages of 450 and/or 600 mg/kg/day).

Pharmacokinetic Evaluation (R990202-PK)

Tenofovir concentrations were determined in plasma and milk from pregnant female rats dosed with TDF by oral gavage for approximately 27 days at 0, 50, 150, 450, or 600 mg/kg/day (Tabulated Summary 2.6.5.7.F, R990202-PK). Blood samples were taken twice, once on Day 7 of presumed gestation (the first day of dosing) and once on Day 20 postpartum (the last day of dosing). Milk samples were collected at approximately 1 hour postdose on Day 11 postpartum.

Results for Day 7 median TFV plasma concentration verses time profiles showed rapid absorption of TFV followed by biexponential elimination for all dose groups. Plasma pharmacokinetic Tmax values varied from 0.25 to 0.67 hours across the dose range. Median Cmax values were 1271, 2525, 3434, and 4710 ng/mL for the 50, 150, 450, and 600 mg/kg/day dose groups, respectively. Median AUCinf values were 3811, 7840, 54,373, and 28,961 ng·h/mL for the 50, 150, 450, and 650 mg/kg/day dose groups, respectively. Median Cmax and AUCinf values showed a general increasing trend with dose that was not proportional, but this may be misleading due to the high inter-subject variability and the short sampling time (12 to 24 hours).

Results for Day 20 median TFV plasma concentration verses time profiles showed rapid absorption of TFV followed by biexponential elimination for all dose groups. The 600 mg/kg/day dose group showed an almost linear decline from 0.67 to 12 hours. Tmax values varied from 0.67 to 1.0 hour across the dose range. Median Cmax values were 1461, 2256, 4933, and 2892 ng/mL for the 50, 150, 450, and 600 mg/kg/day dose groups, respectively. Median AUCinf values were 5883, 11,716, 26,761, and 27,286 ng·h/mL for the 50, 150, 450, and 600 mg/kg/day dose groups, respectively.

Comparison of single dose Day 7 data with steady state Day 20 data revealed overall similar Tmax and apparent terminal t1/2 values. Also, both showed generally increasing values with dose for Cmax and AUC and some apparent accumulation with multiple dosing, although this evaluation is confounded by the high variability and the limited data from the highest dose groups. Results for Day 11 median TFV concentrations from rat milk were 154, 249, 532, and 730 ng/mL for the 50, 150, 450, and 600 mg/kg/day dose groups, respectively; representing 13.5%, 11.0%, 20.8%, and 23.5% of the TFV plasma concentration at similar



times. Although there is a trend towards increasing percent in milk with increasing dose, the data is weak due to the comparison of data from different rats and the different numbers of observations between plasma data (n = 3 to 4) and milk (n = 6 to 11).

In conclusion, all rats in dose groups that received TDF demonstrated exposure based on the measurable levels of TFV in plasma on Days 7 and 20. Both Day 7 and Day 20 postpartum pharmacokinetic rat plasma profiles demonstrated increased exposure of TFV with increasing oral doses of TDF. Day 11 postpartum data confirmed the presence of TFV in rat milk, with the results suggesting that TFV was not concentrated in milk.


2.6.6.6.4.1. Fertility and Early Embryonic Development

The reproductive and developmental NOELs for the individual agents were at exposure levels well above human exposures. With no expected pharmacokinetic or toxicologic interactions with the FTC/RPV/TDF combination, further studies with FTC/RPV/TDF are not considered necessary.

2.6.6.6.4.2. Embryo-Fetal Development

There were no effects on embryo-fetal development in rats or rabbits when FTC, RPV, and TDF were tested individually. No cause for concern has been identified and studies with the FTC/RPV/TDF combination are unlikely to show new effects.

2.6.6.6.4.3. Prenatal and Postnatal Development

Slightly longer oestrous cycles were observed in F1 generation rats after exposure to high doses of FTC and a delay in sexual maturation was observed in F1 generation rats after exposure to high (maternally toxic) doses of TDF. No significant effects were noted for RPV. For all 3 individual agents, NOELs were at exposures above human exposures. As with other reproductive toxicity tests, a repeat of this test with the FTC/RPV/TDF combination is unlikely to add any new information.

2.6.6.6.4.4. Juvenile Animals

No specific studies were conducted with the combination. Use of the FTC/RPV/TDF tablet in young children is not anticipated. Guidance is provided in the prescribing information that the FTC/RPV/TDF tablet should not be administered to children or adolescents.

2.6.6.7. Local Tolerance

2.6.6.7.1. Rilpivirine: Eye Irritation – Bovine Corneal Opacity-Permeability Assay

The ocular irritation potential of TMC278 was assessed in the bovine corneal opacity permeability (BCOP) assay. TMC278 was applied for 4 hours as a 20% w/w suspension in saline to the corneal epithelium (Tabulated Summary 2.6.7.16.A, TMC278-NC202).



An increase in corneal opacity was noted, but no effect on permeability occurred. The score of 32.5 ± 3.0 classified TMC278 as a moderate eye irritant.

2.6.6.7.2. Rilpivirine: Phototoxicity – Balb/c Mouse 3T3 Fibroblast Assay

The in vitro Balb/c mouse 3T3 fibroblast assay was used to assess the phototoxic and cytotoxic potential of TMC278 base. Cells were exposed to TMC278 base dissolved in DMSO in bath concentrations ranging from 0.001 to 2.16 mg/L, in the presence and the absence of UVA radiation. The final concentration of DMSO in Dulbecco’s phosphate-buffered saline was 1% (Tabulated Summary 2.6.7.16.A, TMC278-NC188).

As the highest concentration had no effect on the Neutral Red uptake by these cells in the presence of UVA, TMC278 was considered to be nonphototoxic.

2.6.6.7.3. Rilpivirine: Skin Irritation – In Vivo

The skin irritation potential of TMC278 was evaluated in 3 New Zealand white rabbits. TMC278 (0.5 g moistened with 0.5 mL water on a gauze) was applied to 4 4 cm on the intact clipped skin for 4 hours covered by a semi-occlusive dressing. The skin reactions were scored 1, 24, 48 and 72 hours after removal of the dressing (Tabulated Summary 2.6.7.16.A, TMC278-NC159).

All animals survived and there were no noteworthy clinical observations or effects on body weight. No skin reactions, staining of skin, or corrosive effects were observed. TMC278 was classified as “nonirritant” to rabbit skin.

2.6.6.7.4. Tenofovir DF: A Primary Eye Irritation Study in Rabbits with Tenofovir DF

The potential irritant and/or corrosive effects of TDF (GS-4331-05) were evaluated on the eyes of New Zealand White rabbits (Tabulated Summary 2.6.7.16.B, B990165). Each of 6 rabbits received a 0.0760 g (0.1 mL dose equivalent) dose of the test article in the conjunctival sac of the right eye. Approximately 30 seconds following dosing, the eyes of 3 rabbits were rinsed with sterile water (rinsed group). The remaining animals were not rinsed (no rinse group). The contralateral eye of each animal remained untreated and served as a control. Test and control eyes were examined for signs of irritation for up to 21 days following dosing.

In the no rinse group, exposure to the test article produced corneal opacity in 3/3 test eyes at the 1-hour scoring interval. The corneal injury was confirmed by positive fluorescein dye retention at the 24-hour scoring interval. The corneal opacity did not resolve in any test eyes by study Day 21. Iritis was observed in 3/3 test eyes at the 1-hour scoring interval and resolved completely in all test eyes by study Day 21. Conjunctivitis was noted in 3/3 eyes at the 1-hour scoring interval and resolved completely in 2/3 test eyes by study Day 21. Additional ocular findings included apparent blanching on the nictitating membrane (3/3 test eyes), corneal bulging (3/3 test eyes), corneal neovascularization (3/3 test eyes), raised area(s) on the cornea (1/3 test eyes), and sloughing of the corneal epithelium (1/3 test eyes).



In the rinsed group, exposure to the test article produced corneal opacity in 3/3 test eyes at the 1-hour scoring interval. The corneal injury was confirmed by positive fluorescein dye retention at the 24-hour scoring interval. The corneal opacity resolved in 2/3 test eyes by study Day 21. Iritis was observed in 3/3 test eyes at the 1-hour scoring interval and resolved completely in all test eyes by study Day 21. Conjunctivitis (redness, swelling and discharge) was noted in 3/3 test eyes at the 1-hour scoring interval. The conjunctival irritation resolved completely in 2/3 test eyes by study Day 21. Additional ocular findings included apparent blanching on the nictitating membrane (2/3 test eyes), corneal bulging (1/3 test eyes), raised area(s) on the cornea (1/3 test eyes), and corneal neovascularization (3/3 test eyes).

Based on the no rinse and rinsed groups, TDF (GS-4331-05) is considered to be a very severe irritant to the ocular tissue of the rabbit.

2.6.6.7.5. Tenofovir DF: A Primary Skin Irritation Study in Rabbits with Tenofovir DF

The potential irritant and/or corrosive effects of TDF (GS-4331-05) were evaluated on the skin of New Zealand White rabbits (Tabulated Summary 2.6.7.16.B, B990166). Each of 6 rabbits received a 0.5 g dose of the test article as a single dermal application. The test article was moistened with deionized water to enhance test article contact with the skin. The dose was held in contact with the skin under a semi-occlusive binder for an exposure period of 4 hours. Following the exposure period, the binder was removed and the remaining test article was wiped from the skin using gauze moistened with deionized water followed by dry gauze. Test sites were subsequently examined and scored for dermal irritation for up to 72 hours following patch application.

Exposure to the test article produced very slight erythema on 1/6 test sites at the 1-hour scoring interval. The dermal irritation resolved completely in the affected animal by the 24-hour scoring interval. Under the conditions of this test, TDF (GS-4331-05) is considered to be a slight irritant to the skin of the rabbit. The calculated Primary Irritation Index for the test article was 0.04.


The FTC/RPV/TDF tablet is intended for oral use. No local tolerance studies were conducted for the FTC/RPV/TDF combination.



2.6.6.8. Other Toxicity Studies

2.6.6.8.1. Antigenicity

2.6.6.8.1.1. Rilpivirine: Hypersensitivity – In Vivo

A murine local lymph node assay was conducted to establish the sensitization potential of TMC278 (Tabulated Summary 2.6.7.16.A, TMC278-NC199). The compound was formulated in an acetone:olive oil (4:1) mixture at 5%, 10%, and 35%. Twenty-five microliters of each concentration was applied for 3 consecutive days on the dorsal surface of both ears of 4 female CBA/Ca/Ola/Hsd mice per group. The negative control group received the vehicle. The positive control group was treated with 2.5% streptozocin. Three days later, all animals received an intravenous injection of [3H]methyl thymidine. Five hours later they were killed and the radioactivity in the auricular lymph nodes was measured.

The test to control ratio of radioactivity from thymidine incorporated in the draining lymph node ranged from 0.6 to 1.7, indicating that TMC278 was unlikely to cause delayed-type sensitization.

In view of the absence of indications from repeat dose toxicity studies (see Section 2.6.6.3) that TMC278 might elicit allergic reactions, no specific antigenicity studies have been conducted.

2.6.6.8.1.2. Tenofovir DF: A Dermal Sensitization Study in Guinea Pigs with Tenofovir DF (GS-4331-05) Modified Buehler Design (G990167)

The dermal sensitization potential of TDF was evaluated in Hartley-derived albino guinea pigs (Tabulated Summary 2.6.7.17.C, G990167). Ten male and 10 female guinea pigs were topically treated with 75% w/v TDF (GS-4331-05) in PEG 400, once per week, for 3 consecutive weeks. Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naive) challenge control guinea pigs were topically treated with 50% w/v TDF in PEG 400 and 100% TDF (GS-4331-05). Challenge responses in the test animals were compared with those of the challenge control animals. Following a 7-day rest period, a rechallenge was performed whereby the 20 test animals and 10 previously untreated (naive) rechallenge control guinea pigs were topically treated with 65% w/v TDF in PEG 400. Rechallenge responses in the test animals were compared with those of the rechallenge control animals. A 1-chloro-2,4-dinitrobenzene (DNCB) positive control group consisting of 10 DNCB test and 10 DNCB control guinea pigs was included in this study. The DNCB test animals received 0.1% w/v DNCB in acetone/ethanol for induction and 0.1% and 0.05% w/v DNCB in acetone/ethanol for challenge.

Following challenge with 50% w/v TDF in PEG 400, dermal scores of 0 to were noted in all test and challenge control animals. Group mean dermal scores were noted to be similar in the test animals as compared with the challenge control animals. Following challenge with 100% TDF, dermal scores of 0 to were noted in all test and challenge control animals. Group mean dermal scores were noted to be similar in the test animals as compared with the challenge control animals. Following rechallenge with 65% w/v TDF in PEG 400, dermal



scores of 0 to were noted in all test and rechallenge control animals. Group mean dermal scores were noted to be similar in the test animals as compared with the rechallenge control animals. Following challenge with DNCB, the DNCB test animals were noted to have a substantially stronger dermal response than was observed in the corresponding DNCB control animals. Group mean dermal scores were also noted to be higher in the DNCB test animals as compared to those of the DNCB control animals. Based on the results of this study, TDF is not considered to be a contact sensitizer in guinea pigs. The results of the DNCB positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.

2.6.6.8.1.3. Emtricitabine/Rilpivirine/Tenofovir DF

No specific antigenicity studies have been conducted with FTC. Findings with RPV and TDF have not indicated that antigenicity is of concern. No studies were deemed necessary with the FTC/RPV/TDF combination.

2.6.6.8.2. Immunotoxicity

2.6.6.8.2.1. Emtricitabine: TP-0006 – 28 Day Oral (Gavage) Immunotoxicity Study in Rats

The immunotoxicity of FTC was evaluated in rats given once daily oral doses of FTC for 28 consecutive days (Tabulated Summary 2.6.7.17.A, TOX146). Emtricitabine was suspended in 0.5% methylcellulose and given by gavage to groups of 5 male and 5 female Sprague-Dawley rats at 0, 60, 240, and 1000 mg/kg/day. Following the final dose, each rat was given an intravenous dose of 2 108 sheep RBCs. Five days later, blood samples were collected and the serum was analyzed by ELISA to detect IgM antibodies specific to sheep RBCs. There were no adverse effects of FTC during the dose period. Body weights were unaffected by treatment with FTC. Emtricitabine did not affect the titers to sheep RBCs at any of the doses administered. It was concluded that 1000 mg/kg/day FTC was a no-effect dose for immunotoxic potential.

2.6.6.8.2.2. Rilpivirine

The lack of effects on the PFC-assay in the 1-month rat study (TMC278-TOX5692; see Section 2.6.6.3.2.3) indicated that TMC278 had no immunotoxic potential.


Data from repeat-dose toxicity studies with FTC, RPV, or TDF (hematology, lymphoid organ weights, microscopy of lymphoid tissues, bone marrow cellularity) and some specialized assays (FTC and RPV) did not suggest immunotoxic potential for any of these agents. There were no notable effects of the combination of FTC/TDF on immunophenotyping or NK cell assay values in a 4-week dog study (Tabulated Summary 2.6.7.7.T, TX-164-2004).



2.6.6.8.3. Mechanistic Studies

For RPV, studies were conducted to address the mechanism of action of the effects on the biosynthesis of adrenocortical hormones observed in general toxicity studies in rats and dogs and on the functioning of the pituitary-adrenal axis. For TDF, studies were conducted in rats, woodchucks, and monkeys to evaluate the potential for mitochondrial toxicity. Further, a series of studies were conducted in vitro and in vivo in rats and monkeys with TDF to evaluate bone and renal effects.

2.6.6.8.3.1. Studies to Evaluate Endocrine Effects

2.6.6.8.3.1.1. Rilpivirine: Inhibition of ACTH-stimulated Cortisol Biosynthesis in Primary Cultures of Guinea Pig Adrenal Cells

TMC278 was assessed for its ability to inhibit cortisol biosynthesis in primary cultures of guinea pig adrenal cells (Tabulated Summary 2.6.7.17.B, TMC278-Exp5653). The effects of TMC278 base dissolved in DMSO (final bath concentration 1%) in concentrations ranging from 1 to 10 μM upon the cortisol synthesis stimulated by ACTH1-24 were determined in 2 independent repeats. Etomidate served as positive control.

TMC278 inhibited cortisol synthesis in one experiment with a median inhibitory concentration (IC50) of 6 μM, but showed no significant inhibition in the second experiment. Etomidate rendered an IC50 value of 0.5 nM.

2.6.6.8.3.1.2. Rilpivirine: Evaluation of the Cortisol Biosynthesis in Crude Subcellular Fractions of Dog Adrenal Cortex

The effect of TMC278 base on adrenal corticosteroid biosynthesis was studied in vitro using cell free adrenal gland homogenate (E-fraction) from beagle dog (Tabulated Summary 2.6.7.17.B, TMC278-FK4790). Nominal concentrations of TMC278 ranged from 0.03 to 75 μM (0.011 to 27.75 μg/mL). Etomidate (0.28 μM) served as positive control.

TMC278 at a concentration of 75 μM (27.75 μg/mL) caused 39% inhibition of the metabolism of pregnenolone compared to control. A concentration-dependent increase in progesterone and 17 -hydroxyprogesterone concentrations was noted concomitant with decreases of 11-deoxycorticosterone, 11-deoxycortisol, and corticosterone concentrations. Etomidate showed a 38% inhibition of the corticosteroid synthesis at 0.28 μM.

2.6.6.8.3.1.3. Rilpivirine: Single Dose Oral Endocrinology Study in Dogs

TMC278 base dissolved in PEG400 + CA was administered once, by oral gavage, at 0, 20, or 80 mg/kg in a dose volume of up to 10 mL/kg to groups of 6 male beagle dogs (Tabulated Summary 2.6.7.17.B, TMC278-Exp5627). Mortality, clinical observations, body weight, and body weight gain were evaluated. Cortisol, ACTH, and aldosterone levels were measured (1 and 6 days after dosing). Pituitary-adrenocortical function was assessed by a challenge with corticotrophin releasing factor (CRF) (1 and 6 days after dosing). Toxicokinetic evaluation on blood samples taken on the day of dosing was also included.



There were no mortalities associated with TMC278. There were no relevant clinical signs, or effects on body weight or body weight gain. No relevant changes in plasma cortisol, aldosterone, or ACTH were recorded. At the CRF challenge, no relevant changes were seen in plasma cortisol levels. Systemic exposure expressed as Cmax and AUC values are given in Table 28 below.

Table 28. Exposure of TMC278 (Cmax and AUC) in a Single Dose Oral Endocrinology Study in Dogs with TMC278 Base

Cmax (μg/mL) AUCinf (μg.h/mL) Dose

(mg/kg/day) Sampling Day M (n = 6)

20 Day 1 2.8 87

80 Day 1 2.8 105

M: males

2.6.6.8.3.2. Evaluations of Mitochondrial Toxicity

2.6.6.8.3.2.1. Emtricitabine

In 1-month and 3-month oral toxicity studies in cynomolgus monkeys, no evidence of mitochondrial-related hepatic, cardiac or skeletal muscle toxicity was detected after examination by transmission electron microscopy (Tabulated Summaries 2.6.7.7.U and 2.6.7.7.V, TOX600 and TOX627, respectively).

2.6.6.8.3.2.2. Rilpivirine

Refer to Section 2.6.2.2.6.2 for a summary of in vitro studies of mitochondrial effects.

2.6.6.8.3.2.3. Tenofovir DF

In chronic studies (42-week rat or dog studies), no evidence of mitochondrial-related hepatic, hematologic, cardiac, pancreatic, or skeletal muscle toxicity was detected (Tabulated Summaries 2.6.7.7.L and 2.6.7.7.S, 97-TOX-4331-002 and 97-TOX-4331-001, respectively). The potential to cause mitochondrial toxicity in vivo was evaluated in rats, woodchucks, and monkeys (see Section 2.6.6.8.3.3.6, Tabulated Summaries 2.6.7.17.C, P2000078). These studies used specific biochemical and morphologic evaluations to determine potential in vivo effects of TDF on mitochondrial function and mitochondrial DNA incorporation (see below).

A 28-Day Oral Repeat Dose Toxicity Study to Evaluate the Toxicity of Adefovir Dipivoxil (GS-0840), Tenofovir DF (GS-4331-05) or Adefovir Dipivoxil in combination with Tenofovir DF in Male Sprague Dawley Rats (R2000096)

In this non-GLP study, Sprague Dawley rats (6 males/group) were administered TDF (300 mg/kg/day), adefovir dipivoxil (40 mg/kg/day), or an admixture of TDF and adefovir



dipivoxil for 28 days to evaluate and compare general toxicity and assess markers of mitochondrial toxicity (Tabulated Summary 2.6.7.17.C, R2000096). This summary focuses on the findings in the TDF alone group. There were few clinical signs and no changes in body weight in the TDF group. Small elevations in BUN, AST, and ALT were observed, as well as increased urinary calcium excretion and decreased urinary phosphorus excretion. The group treated with TDF alone had minimal renal cortical tubular karyomegaly, dilatation of the ileum and colon, and thickening of the duodenal mucosa. There were no indications of mitochondrial dysfunction as measured by changes in serum lactate or cytochrome c oxidase, citrate synthase, or mtDNA content in liver, kidney, or skeletal muscle.

Tenofovir DF: A 90 -Day Repeat Dose Oral Toxicity Study of Tenofovir DF (GS-4331-05) in Woodchucks (W2000042)

The woodchuck model was chosen to evaluate the potential for nucleoside-induced mitochondrial toxicity based on published reports describing the toxicological effects of fialuridine and other nucleosides in this model {6124}, {1790}, {6120}. The object of this study was to assess the toxicity of TDF in woodchucks, including delayed mitochondrial toxicity, during a 90-day treatment period (Tabulated Summary 2.6.7.17.C, W2000042). Six normal adult woodchucks received daily oral doses of 15 mg/kg of TDF, 6 woodchucks received daily oral doses of 50 mg/kg of TDF, and 6 control animals received vehicle, orally each day, as placebo.

There were no mortalities during the study and no clear treatment-related clinical signs. There were no treatment-related changes in food intake, body weights, and organ weights. There were no important changes in hematology, clinical chemistry (including serum lactate), coagulation, or urinalysis analytes. There were no treatment-related gross or microscopic alterations. The mitochondrial evaluation included assessment of cytochrome c oxidase, citrate synthase, and mitochondrial DNA content of liver, kidney, skeletal, and cardiac muscle. There was no evidence of mitochondrial injury. The NOEL for this study was considered to be 50 mg/kg/day.



Pharmacokinetic Evaluation (W2000042-PK):

Blood samples were collected from each animal at 0.75 and 24 hours postdose on study Days 0 and 88 (Tabulated Summary 2.6.7.17.C). Plasma samples were analyzed by LC/MS/MS for TFV. All animals showed sustained plasma exposure to TFV over the course of the study. Similar TFV plasma concentrations were observed in male and female animals. Following the first dose of TDF (Day 0), TFV plasma concentrations at 0.75 hour (approximating Cmax) appeared to increase proportionally with dose (292 127 and 988 682 ng/mL at 15 and 50 mg/kg/day, respectively). Tenofovir plasma concentrations obtained at 24 hours were similar in the 2 treated groups, 21.9 21.9 (15 mg/kg/day) and 21.7 8.77 (50 mg/kg/day) ng/mL.

Following repeated oral dosing of TDF, increases of approximately 4-fold in TFV plasma concentrations were observed at 24 hours postdose (for both dose groups). This data suggests accumulation of TFV in plasma that was not predicted by single dose studies in these same animals. The reason for this accumulation cannot be discerned from this study.

2.6.6.8.3.2.4. Emtricitabine/Rilpivirine/Tenofovir DF

In a variety of in vitro and in vivo studies, FTC and TFV/TDF have shown a low potential for mitochondrial toxicity. In vitro studies to examine the potential mitochondrial effects of combinations of NRTIs, including FTC in combination with TFV have shown no adverse effects on various mitochondrial parameters (TX-104-2001). In an in vitro study with RPV, there were no inhibitory effects on human polymerases, , , or (Section 2.6.2.2.6.2), suggesting a low potential for mitochondrial toxicity. However, as mitochondrial toxicity is generally less relevant for NNRTIs than NRTIs, and as RPV is not anticipated to significantly increase the exposure of FTC or TFV, the potential for exacerbating mitochondrial toxicity is low.

2.6.6.8.3.3. Studies to Evaluate Bone or Kidney Toxicity

2.6.6.8.3.3.1. Tenofovir DF: Human Osteoblast Calcium Deposition In Vitro

The objective of the study was to determine if prolonged exposure of human osteoblast-like (NHOst) cells to TFV was cytotoxic or altered their function to deposit mineralized calcium (Tabulated Summary 2.6.7.17.C, V2000122). NHOst cells were maintained as either undifferentiated (cultured in OGM medium) or differentiated cells (cultured in Diff. A medium) in 96-well tissue culture treated plates, in the presence or absence of TFV. NHOst cells require culture in differentiating media (Diff. A) in order to deposit mineralized calcium in vitro. Tenofovir was added to differentiated cells only. NHOst cells cultured in OGM medium served as non-calcium depositing control groups. NHOst cells were exposed to TFV at a concentration of 10 g/mL, 3 times the plasma concentration area under the curve (AUCss) and 27 times the Cmax for the human dose (300 mg/day), and maintained with twice weekly medium changes for 3-weeks. The NHOst cells were processed using the Von Kossa mineralized calcium staining method (5% silver nitrate, 0.5% hydroquinone, and



5% sodium thiosulfate) for assessment of calcium deposition. Prolonged exposure to TFV did not cause cytotoxicity nor did it alter calcium deposition.

2.6.6.8.3.3.2. Tenofovir DF: A 3-Day Oral Repeat Dose Study to Evaluate Serum and Urine Phosphorous Levels in Tenofovir DF (GS-4331-05)-Treated Male Sprague-Dawley Rats Supplemented, Following the Final Dose, with Intraperitoneal or Oral Phosphate

Previous studies have shown that TDF administered to rats by oral gavage causes an acute, marked hypophosphaturia with minimal effect on the serum phosphorus concentration. The objective of this study was to evaluate the serum phosphorus concentration and urinary phosphorus excretion in male Sprague-Dawley rats (2 groups, 6M/group) treated with TDF, 400 mg/kg/day, administered by oral gavage daily, for 3 consecutive days; and then given a single dose of sodium phosphate, 280 mg phosphate/kg, by either an IP injection or by oral gavage immediately following the third TDF dose (Tabulated Summary 2.6.7.17.C, R2000095). Control groups (2 groups, 6M/group) received vehicle by oral gavage and sodium phosphate, 280 mg phosphate/kg, by either an IP injection or by oral gavage.

There were no deaths or abnormal clinical observations. Treatment of rats with 400 mg/kg/day of TDF administered by oral gavage daily for 2 days caused severe hypophosphaturia and a slight reduction of the serum phosphorus concentrations. The administration of a single dose of 280 mg phosphate/kg by either oral gavage or IP injection normalized the serum phosphorus concentration and increased urinary phosphorus excretion in all groups. Notably, urinary phosphorus excretion was attenuated in the group that received the phosphate by oral gavage.

2.6.6.8.3.3.3. Tenofovir DF: A 3-Day Oral or Intravenous Repeat Dose Study to Evaluate Serum and Urine Phosphate Concentrations in Male Sprague-Dawley Rats Treated with Tenofovir (GS-1278) or Tenofovir DF (GS-4331-05) and Supplemented with Oral Phosphate

The objective of the study was to evaluate the effects of TDF (400 mg/kg/day) administered daily by oral gavage for 3 days or TFV administered daily by oral gavage (180 mg/kg/day) for 3 days or by daily intravenous bolus injection (50 mg/kg/day) for 3 days on the serum phosphorus concentration and urinary phosphorus excretion in male rats (6/group), and the subsequent effects of sodium phosphate (280 mg phosphate/kg) administered by oral gavage immediately following the third dose of test article or vehicle control (Tabulated Summary 2.6.7.17.C, R2000099). The control group received vehicle (50 mM CA).

The administration of TDF at a dose of 400 mg/kg/day for 3 days caused marked hypophosphaturia and a slight reduction of the serum phosphorus concentration. The intravenous bolus injection administration of TFV caused a slight reduction of the serum phosphorus concentration, but did not alter urinary phosphorus excretion. The administration of TFV by oral gavage did not alter the serum phosphorus concentration or urinary phosphorus excretion. Four hours after the administration of phosphate by oral gavage, all groups had increased urinary phosphorus excretion of similar magnitude, except the TDF



group which had an increase that was only about one-third of the other groups. The serum phosphorus normalized in the TDF- and intravenous TFV -treated groups following phosphate supplementation. The findings suggest that the oral gavage administration of the prodrug of TFV, TDF, and not TFV itself, impaired intestinal phosphate absorption with a response by the kidneys to retain phosphate. Phosphate supplementation by oral gavage appeared to overcome the impairment resulting in increased urinary phosphorus excretion. Neither TFV administered orally or intravenously altered urinary phosphorus excretion before or after phosphate supplementation.

2.6.6.8.3.3.4. Tenofovir DF: A 6-Day Repeat Dose Oral Exploratory Study of Tenofovir DF in Male Sprague-Dawley Rats

The objective of this study was to examine the effect of daily treatment of TDF on serum or plasma calcium and phosphorus concentrations; urinary excretion of calcium, phosphorus, cAMP, and NAG; and the biochemical markers of bone remodeling in rats (Tabulated Summary 2.6.7.17.C, R2000043). Male Sprague Dawley rats were administered 0 or 400 mg/kg/day of TDF by oral gavage for 6 days. Treatment resulted in a marked, persistent hypophosphaturia. There was an initial hypocalciuria, followed by values that approximated the mean control values on Days 3, 4, and 5. The plasma ionized calcium concentration was decreased at 2 hours and increased at 24 hours after TDF treatment and the total calcium concentration was slightly increased on Day 6. The plasma phosphorus concentration was increased at 2 hours and decreased at 24 hours after TDF treatment and approximated the control value on Day 6. The urinary cAMP approximated control values and the urinary NAG were reduced at all time points. The serum 25-vitamin D3 concentration was decreased, and the 1,25-vitamin D3 concentration was increased for the TDF-treated group. The PTH concentration for the TDF-treated group approximated the control value. The hypophosphaturia was considered to be most likely due to impaired intestinal absorption of phosphate. Smaller changes in the serum phosphorus and PTH concentrations were considered to be a response to mobilize additional phosphate.

2.6.6.8.3.3.5. Tenofovir DF: A 28-Day Study to Evaluate the Effects of Tenofovir Disoproxil Fumarate (Tenfovir DF) on Bone Following Daily Administration by Gavage in the Sprague-Dawley Rat

The objective of this study was to evaluate the effects of TDF on bone determined by measurement of biochemical markers of bone turnover, histomorphometry, and densitometry in male Sprague-Dawley rats (10/group) following oral gavage administration of 0 (vehicle), 40, and 400 mg/kg/day for 28 days (Tabulated Summary 2.6.7.17.C, R2000036). Bone densitometry measurements were performed on the right femur (ex vivo) for all main study animals. Static and dynamic histomorphometry of the proximal metaphysis of the right tibia (ex vivo) was performed on all main study animals.



The administration of TDF at dose levels of 40 and 400 mg/kg/day to male rats for 28 days resulted in dose-related osteopenia, and bone mineral content and bone mineral density, as determined by histomorphometry and bone densitometry, respectively. Microscopic indices of bone formation and mineralization were considered to be unaffected by the treatment with TDF. There were no histologic lesions in the decalcified sections of distal femur from either TDF-treated group. Renal cortical tubular karyomegaly was observed in the 400-mg/kg/day group.


Concentrations of TFV were determined in plasma samples obtained during a 28-day repeated dose toxicity study after oral gavage of TDF in male Sprague-Dawley rats (Tabulated Summary 2.6.5.4.CC, R2000036-PK). Blood samples (n = 3/time point) were obtained on Days 1, 13, and 28. Time points included predose and 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 12, and 24 hours postdose. An additional sample was collected at 48 hours for the 400-mg/kg/day dose group. Samples were processed for plasma and concentrations of TFV in plasma were determined using a validated LC-MS assay. Plasma concentration values were subjected to pharmacokinetic analysis using noncompartmental methods. Following the first oral dose in rats, concentrations of TFV in plasma reached mean peak values of 1.06 and 3.17 g/mL at the 40- and 400-mg/kg/day dose levels, respectively; with Tmax values between 0.25 to 0.5 hours, respectively. AUCinf values on Day 1 were 2.55 and 9.67 g·h/mL for the 40- and 400-mg/kg/day dose levels, respectively. However, due to the large percentage of extrapolated AUC (41%) for the 400-mg/kg/day dose group, only limited conclusions should be drawn from the pharmacokinetic parameters for this cohort. On Day 28, the AUC0 t values were 3.13 and 19.6 g·h/mL for the 40- and 400-mg/kg/day dose cohorts, respectively. These values are similar to those observed in other repeat-dose studies of TDF in rats. Following oral administration of TDF on Day 1, Cmax deviated from dose proportionality over the range of 40 to 400 mg/kg/day, suggesting decreased absorption at the higher dose. Repeated administration of TDF over a 28-day period resulted in only minor changes in pharmacokinetics relative to Day 1.

2.6.6.8.3.3.6. Tenofovir DF: A 56-Day Study of Tenfovir DF Administrered Orally and of PMPA Administered by Subcutaneous Injection to Rhesus Monkeys (P2000078)

The primary objective of this study was to assess the general toxicity of TDF and specifically to determine the effects of TDF on calcium and phosphorus homeostasis and on bone remodeling (as assessed by biochemical markers) when administered to Rhesus monkeys for at least 28 days (Tabulated Summary 2.6.7.17.C, P2000078). Tenofovir was also evaluated. Dose groups with decreased serum phosphorus values and/or altered urine phosphorus values following 1 month of TDF and/or TFV administration received oral phosphate supplementation for the later part of the study (Days 29 to 56) to determine the effectiveness of supplementation.



TDF was administered orally by nasogastric gavage at doses of 0 (vehicle), 30, 250, or 600 mg/kg/day (3/sex/group) for 56 consecutive days. Tenofovir was administered at a dose of 30 mg/kg/day for 56 consecutive days by subcutaneous injection (3/sex/group). Due to the poor clinical condition of some of the animals in the 600 mg/kg/day oral TDF group (see below), this group was terminated early (i.e., on Days 20 to 23). On Day 29, animals in the remaining groups received 500 mg/kg/day phosphorus orally. However this initial dose level was associated with emesis and diarrhea during the first 2 days and hence the dose was reduced to a level that provided 125 mg/kg/day phosphorus thereafter.

During the first 3 weeks of the study, 2 animals that received 600 mg/kg/day developed severe renal toxicity that was associated with clinical signs of poor condition and alterations in clinical pathology parameters (elevations in serum BUN and creatinine concentrations). Consequently, the entire group was terminated on Days 20 to 23. A lesser degree of kidney dysfunction was evident in 1 other monkey treated with the highest dose level of TDF and in 1 animal that received TFV by subcutaneous injection at a dose level of 30 mg/kg/day. Microscopic examination revealed histopathologic changes in the kidneys of the majority of animals that received either 250 or 600 mg/kg/day of TDF or 30 mg/kg/day of TFV. The most severe degenerative lesions, involving diffuse tubular necrosis, occurred at a low incidence and were correlated with the magnitude of clinical pathology alterations in individual animals. There were also clinical signs of GI disturbance (i.e., an increased incidence of emesis and unformed stools) in animals that received the 250 and 600 mg/kg/day dose levels of TDF, the incidence of which was dose-dependent.

The daily oral administration of TDF or subcutaneous administration of TFV caused a reduction of the mean serum phosphorus concentration. The lack of concurrent hyperphosphaturia in most animals suggested an impairment of intestinal phosphate absorption that was corrected by oral phosphate supplementation. However, some animals in the 600 mg/kg/day TDF and 30 mg/kg/day TFV groups did have hyperphosphaturia, which at least in part contributed to the altered phosphate homeostasis. Hypercalciuria developed in both the TDF- and TFV-treated groups, suggesting an effect, either direct or indirect, on the ability of the renal tubule to retain calcium.

Gross necropsy findings and organ weight changes were generally consistent with the kidney as the primary target organ, with some indications of secondary alterations in the liver and thymus, most commonly in animals that received the high dose level of TDF.

The only other histologic findings that were regarded as most likely a direct effect of the test articles were testicular lesions of animals from all treatment groups. These were characterized by the presence of spermatidic multinucleated giant cells within some seminiferous tubules accompanied by decreased numbers of spermatozoa. Unequivocal discernment of the prevalence, dose-dependence, and biological significance of this alteration was hampered by the small number of animals on study with sexually mature testes.



There were no consistent changes in the mean serum values for 25-hydroxy vitamin D3, 1,25-dihydroxy vitamin D3, bone-derived ALP, and urinary N-telopeptide. There was a reduction in mean PTH concentration during the first 3 weeks of treatment in individual animals that received 250 and 600 mg/kg/day of TDF orally (except 2 monkeys with severe renal failure) or 30 mg/kg/day of TFV subcutaneously. However, there was no characteristic decrease in serum calcium or increase in serum phosphorus levels. Importantly, there was no alteration in bone morphology that could be clearly ascribed to the test articles.

Biochemical analysis and electron microscopic examination of sections of the kidney, liver, cardiac muscle, and skeletal muscle from animals that received TDF at 0, 30, and 250 mg/kg/day indicated no potential to cause mitochondrial injury at dose levels up to 250 mg/kg/day.

Pharmacokinetic Evaluation (P2000078-PK)

The toxicokinetic portion of study P2000078 was designed to demonstrate plasma and peripheral blood mononuclear cell (PBMC) exposure to TFV following oral administration of TDF or subcutaneous administration of TFV in monkeys (Tabulated Summary 2.6.5.4.JJ,P2000078-PK). Blood samples were collected predose and 0.5, 1, 2, 6, 12, and 24 hours postdosing on study Days 1 and 27 (samples from Group 4 animals sacrificed early due to toxicity were collected on Day 20 to 23), and at predose and at 0.5, 1, 2, 6, 12, 24, and 48 hours following the Day 55 dose. Plasma samples were analyzed by LC/MS/MS for TFV. PBMCs were collected mid-study and at the end of the study to determine intracellular concentrations of TFV.

All monkeys showed sustained exposure to TFV over the course of the study. Increasing oral doses of TDF led to increased, though not dose proportional, Cmax and AUC values. This was consistent with previous observations that the bioavailability of TFV from oral TDF decreases with increasing dose. There were no significant differences in the pharmacokinetics of TFV between male and female monkeys in any dose group.

The mean AUC observed at steady state for the 30 mg/kg/day oral TDF dose group was 4.05 0.958 g·h/ml, which was approximately 33% greater then the AUC observed in humans following 300 mg doses of TDF. Similar, although slightly lower, AUC values were observed in monkeys administered 600 mg/kg/day oral TDF (56.3 22.1 g·h/ml) as compared to 30 mg/kg/day subcutaneous TFV (62.3 g·h/mL), when comparing values from animals without clinical signs of toxicity. This could suggest that the renal toxicity was more closely related to TFV AUC or time above threshold than to Cmax (which was significantly greater following subcutaneous doses).

In general, PBMCs showed exposure to TFV for the 250 and 600 mg/kg/day oral TDF doses and the 30 mg/kg/day subcutaneous TFV dose. Variability in the data precluded any assessments of trends.




Mechanistic studies were conducted with RPV to address effects on adrenocortical hormones and on the functioning of the pituitary/adrenal axis. Given that neither FTC nor TDF have shown any potential for similar endocrinological changes, it is doubtful that combination dosing would alter the toxicity profile.

In vivo studies to address potential mitochondrial effects were conducted with FTC and TDF. In vitro studies to examine mitochondrial effects of combinations of NRTIs, including FTC in combination with TFV, have shown no adverse effects on various mitochondrial parameters. It is doubtful that combination dosing with the NNRTI RPV and the 2 NRTIs, FTC and TDF, would alter the toxicity profile. Prescribing information contains a class labeling for NRTIs to alert physicians to possible mitochondrial toxicity.

Mechanistic studies to address bone and renal effects were conducted only with TDF. Given the lack of effect on these organs by very high doses of FTC, and that kidney effects (nephropathy) with RPV have been observed in mice and dogs only at high doses, it is doubtful that combination dosing would alter the toxicity profile. Prescribing information contains appropriate text to alert physicians to possible renal or bone effects.

2.6.6.8.4. Dependence

No specific studies have been conducted to evaluate dependency. Nucleoside analogs, as a class, have no known properties that would suggest development of dependence. There was no evidence of development of dependence in preclinical studies with TDF, RPV, or FTC. Consequently, dependency studies were not considered warranted.

2.6.6.8.5. Studies on Metabolites

In view of the absence of unique human metabolites of FTC, RPV, or TDF, and the absence of structural alerts in human metabolites identified (see Module 2.6.4, Section 2.6.4.5), no specific safety studies on FTC, RPV, or TDF metabolites have been conducted.

2.6.6.8.6. Studies on Impurities

2.6.6.8.6.1. Emtricitabine: A 1-Month Mouse Oral Qualification Study of TP-0006 and TP-0296 (Degradant)

The toxicity of FTC in combination with 2% (by weight) TP-0296 (FTU, degradant) when administered orally by gavage to mice for 28 days was evaluated in order to qualify the degradant, TP-0296 (Tabulated Summary 2.6.7.17.A, TOX153). Four groups of mice (10/sex/group) were given 0.5% aqueous methylcellulose vehicle or 50/1, 150/3, or 450/9 mg/kg/day of the combination of FTC/TP-0296, respectively. Each dose level represented the desired FTC dosage in combination with 2% (by weight) dosage of TP-0296. The standard toxicological endpoints were recorded at regular intervals during the study.



All mice survived to the scheduled necropsy. There were no observations related to treatment with the combination of FTC and TP-0296 at any dose level. Body weight, food consumption, hematology, serum biochemistry values, and organ weights were unaffected by administration of the test article combination at any dose level. There were no gross or microscopic findings attributed to the test article combination. The NOEL for oral administration of FTC in combination with TP-0296 was 450/9 mg/kg/day FTC/TP-0296, the highest dose combination tested.

2.6.6.8.6.2. Emtricitabine: A 1-Month Oral Qualification Study of TP-0006 Produced by the Process in CD1 Mice

The objective of the study was to evaluate the toxicity of FTC produced by the Process when administered orally by gavage to mice for a minimum of 28 days to qualify potential impurities and/or degradants in FTC produced by this process (Tabulated Summary 2.6.7.17.A, TX-162-2001). The test article, FTC (GS-9019) in the vehicle, 0.5% aqueous methylcellulose, was administered orally by gavage once daily for a minimum of 28 consecutive days to 3 groups (Groups 2 to 4) of Crl:CD-1®(ICR) BR mice. Dosage levels were 50, 150, and 450 mg/kg/day of the FTC test article. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 10 mL/kg for all groups. Each group consisted of 10 animals/sex.

All animals were observed BID for mortality and moribundity. Clinical examinations were performed daily and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Clinical pathology evaluations (hematology or serum chemistry) were performed for all mice at the scheduled necropsy (study Week 4). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals in the control and 450-mg/kg/day groups; gross lesions were examined microscopically for all animals.

All animals survived to the scheduled necropsy. There were no test article-related clinical observations. No test article-related effects on body weights, food consumption, hematology, serum chemistry parameters, and organ weights were noted. There were no test article-related macroscopic or microscopic findings at any dose level of the FTC test article.

Based on the results of this study, the NOEL for oral (gavage) administration of the FTC test article (produced by the chemistry method of synthesis) to mice for 28 consecutive days was 450 mg/kg/day, the highest dose tested.

2.6.6.8.6.3. Emtricitabine: Bacterial Reverse Mutation Assay of TP-0006 to Qualify Degradant TP-0296

Emtricitabine in combination with 1% (by weight) TP-0296 (FTU, degradant), was tested in the Bacterial Reverse Mutation Assay to qualify degradant TP-0296 using Salmonellatyphimurium tester strains TA98, TA 100, TA 1535, and TA 1537 and Escherichia coli tester strain WP2uvrA in the presence and absence of S9 metabolic activation (Tabulated Summary 2.6.7.17.A, TOX151). The dose solutions were prepared separately in distilled



water and both test articles were soluble at all concentrations used in the study. The assay was performed in 2 phases, using the plate incorporation method. The first phase, an initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article combination.

In the initial toxicity-mutation assay, dose levels of FTC tested were 1.5, 5.0, 15, 50, 150, 500, 1500, and 5000 g/plate, and the highest dose of TP-0296 tested was 50 g/plate. No toxicity and no positive mutagenic response were observed at any dose. Based on these findings, the maximum doses plated in the confirmatory mutagenicity assay were 5000 g/plate for FTC and 50 g/plate for TP-0296.

In the confirmatory mutagenicity assay, no positive mutagenic response was observed. The dose levels of FTC tested were 50, 150, 500, 1500, and 5000 g/plate. The dose levels of TP-0296 tested were 0.50, 1.5, 5.0, 15, and 50 g/plate. Neither precipitate nor toxicity was observed. The combination of FTC and 1% TP-0296 was concluded to be negative.

2.6.6.8.6.4. Emtricitabine: In Vitro Mammalian Chromosome Aberration Study of TP-0006 to Qualify Degradant TP-0296

The chromosome aberration assay was used to evaluate the clastogenic potential of the combination of FTC and 1% TP-0296 (FTU, degradant) (Tabulated Summary 2.6.7.17.A, TOX152). This GLP-compliant study of FTC was conducted to qualify TP-0296 using Chinese Hamster ovary (CHO) cells in the absence and presence of S9 metabolic activation. Both FTC and TP-0296 were soluble in water at all concentrations used in the study.

Based on the lack of cytotoxicity in a preliminary study, doses of FTC chosen for the chromosome aberration assay ranged from 313 to 5000 g/mL for both the activated and nonmetabolically activated treatment groups. The cells were treated for 4 and 20 hours in the nonactivated test system and for 4 hours in the S9 activated test system. All cells were harvested 20 hours after treatment initiation. The percentage of cells with structural or numerical aberrations in the FTC/TP-0296 groups was not significantly increased above that of the solvent control at any dose level (p 0.056, Fisher’s exact test).

Based on the results of this study, it was concluded that FTC with 1% TP-0296 was negative for the induction of structural and numerical chromosome aberrations in CHO cells.

2.6.6.8.6.5. Rilpivirine: In Vitro Bacterial Reverse Mutation (Ames) Test with and

and (drug substance impurities, Figure 2), and TMC278 were dissolved in DMSO (Tabulated Summaries 2.6.7.17.B, TMC278-NC308 and TMC278-NC309). Each impurity was spiked at 4% by mixing a solution of the impurity with a solution of TMC278 at a ratio of 4:96. Each spiked solution was tested separately in 2 independent repeats of a bacterial reverse mutation assay (Ames test) with 5 histidine-requiring strains of Salmonella






2.6.6.8.6.11. Rilpivirine: In Vitro Bacterial Reverse Mutation (Ames) Test with

(trace impurity, Figure 4) dissolved in DMSO was evaluated in a bacterial reverse mutation study (Ames test) (Tabulated Summary 2.6.7.17.B, TMC278-1646_0015299) with histidine-requiring Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537. In each independent repeat, 7 concentrations ranging from 78.13 to 5000 g per plate were tested, in the absence or the presence of metabolic activation (Aroclor-induced rat liver S9 mix).

The first test showed no increased frequency of mutations in any of the tested strains, in the presence or the absence of rat liver S9-mix. The second test showed the same results as the first test, except for TA98 in the presence of S6-mix. Almost all concentrations of showed a slightly more than 2-fold increase of the frequency of mutations compared to vehicle control, which showed a low mutation frequency. A third test with TA98 in the presence of S9-mix showed no increased frequency of mutations up to 5000 μg/plate. It was concluded that has no mutagenic potential.

2.6.6.8.6.12. Tenofovir DF: 14-Day Oral Toxicity Study Comparing Tenofovir DF and Degraded Tenofovir DF in Sprague-Dawley Rats

This study was conducted to determine if there are unexpected toxicologic effects of degraded products of TDF following conditions of accelerated degradation in Sprague-Dawley rats dosed via oral gavage for 14 consecutive days (Tabulated Summary 2.6.7.17.C, R2000081). The study consisted of 6 treatment groups and 1 control group, each consisting of 10 CD® (Crl: CD® [SD] IGS BR) rats per sex. The dose levels were 30, 100, and 300 mg/kg, respectively, for both TDF (Groups 2 to 4) and degraded TDF (Groups 5 to 7). The control group (Group 1) received vehicle only. The vehicle was carboxymethylcellulose 0.5% (w/w), benzyl alcohol, 0.9% (w/w), polysorbate 20, 0.5% (w/w), and Sterile Water for Injection USP. The control or test article was administered once daily by oral gavage for 14 consecutive days at a dose volume of 10 mL/kg. Criteria evaluated for treatment effect included mortality, clinical signs, body weights, and food consumption, as well as various hematological, clinical chemistry, urinalysis, organ weight, macroscopic, and microscopic endpoints. In addition, samples for toxicokinetic analysis were collected at designated time points from 6 additional treated groups (Groups 8 to 13), designated as toxicokinetic groups (each consisting of 6 animals/sex).

No treatment-related findings were observed following 14 days of oral gavage dosing of either TDF (Lot No. ) or degraded TDF (Lot No. ) at doses up to 300 mg/kg/day to rats. No differences between treatment with either TDF or degraded TDF were noted.

不純物F*

不純物F*

不純物F*

不純物F*





The toxicokinetic portion of study R2000081 was designed to determine if there were any unexpected toxicologic effects of degraded products of TDF in rats dosed daily by oral gavage for 14 consecutive days (Tabulated Summary 2.6.7.17.C). Conducted under GLP conditions, 6 treatment groups consisted of 6 animals per group per sex. The dose levels were 30, 100, and 300 mg/kg/day, respectively, for both TDF and degraded TDF. Blood samples were obtained prior to dosing and at 0.17, 2, 4, 8, 12, 24 (prior to the Day 2 dose), and 48 hours (Day 14 only) postdose on Days 1 and 14. Plasma samples were assayed, and concentrations of TFV were determined using an HPLC method with mass spectrometric detection. Tenofovir plasma concentration values were subjected to pharmacokinetic analysis using noncompartmental methods.

No statistically significant effects due to gender or between treatments were observed; p = 0.3994 and 0.01018 on Days 1 and 14, respectively. On Day 1, mean values (male and female data combined) for Cmax were 0.25, 0.67, and 0.56 g/mL for the 30, 100, and 300 mg/kg/day TDF doses, respectively. Similarly, for the degraded TDF doses, the mean values for Cmax were 0.25, 0.48 and 0.67 g/mL for the 30, 100, and 300 mg/kg/day doses. The mean AUC were 2.0, 4.3 and 8.6 g·h/mL for the 30, 100, and 300 mg/kg/day TDF doses, respectively. For the degraded TDF doses, similar mean values for AUC were 2.1, 4.8, and 9.8 g·h/ml for the 30, 100, and 300 mg/kg/day doses.

On Day 14, the mean values for Cmax among animals were 0.29, 0.37, and 0.71 g/mL for the 30, 100, and 300 mg/kg/day TDF doses, respectively. Similarly, for degraded TDF doses, the mean values for Cmax were 0.28, 0.51, and 0.86 g/ml for the 30, 100, and 300 mg/kg/day doses. The mean AUC were 2.1, 4.6, and 9.5 g·h/mL for the 30, 100, and 300 mg/kg/day TDF doses, and 2.0, 4.4, and 10 g·h/ml for the degraded TDF.

When both Cmax and AUC were dosed normalized for each treatment, the slopes did not show any statistically significant differences from zero, indicating that Cmax and AUC increased in a dose proportional manner. The close agreement in dose dependent parameters such as Cmax and AUC between TDF and degraded TDF was consistent with a small extent of degradation ( 10%) that has occurred in this test article.

2.6.6.8.6.13. Emtricitabine/Tenofovir DF: Degraded Tablets

A study to investigate the potential toxicity of nondegraded and degraded FTC/TDF tablets following daily oral administration to the rat for a minimum of 14 days was performed. The results of this study are described in Section 2.6.6.3.



2.6.6.8.7. Qualification of Impurities

The impurities and degradation products present in FTC, RPV, TDF, and FTC/TDF tablets have been qualified through toxicology studies which employed drug substance from normal productions batches, laboratory scale batches with enhanced levels of impurities, and samples subjected to forced degradation conditions (high heat and humidity). There are no unique impurities or degradation products present in the FTC/RPV/TDF tablets that require qualification (Module 3.2.P.5.5, Characterization of Impurities [FTC/RPV/TDF Tablets] and Module 3.2.P.5.6, Justification of Specifications [FTC/RPV/TDF Tablets]).

2.6.6.8.7.1. Qualification of Emtricitabine Related Impurities and Degradation Products

The predominant impurities that are observed in FTC are , , , and (the which is ).

These impurities are all closely related to FTC and may be bioavailable. A potential impurity not commonly observed but present in test samples is ( ). is a degradation product of FTC. may

, thereby creating ‘ ’. The 2 observed of are GS-9237 and GS-492127. These impurities have been studies in toxicology studies and were present in the clinical trial materials. A summary of the qualification for these impurities and degradation products is shown in (Table 29). The qualified level is calculated as:

DoseHumanWeight Humanx

100NOELx LevelObserved (%) Qualified

The observed level and the NOEL or NOAEL are listed in the table. The weight used for this calculation was 60 kg and the dose is 200 mg FTC per day.

不純物FA* 不純物FB*

不純物FC* 不純物FD*

不純物FF*不純物FE* 不純物FE*

不純物FF*

不純物FF* 不純物FF*




Table 29. Qualification Summary for FTC Related Impurities and Degradation Products

Impurity

Impurity Maximum

Observed in Toxicological Studies (%)

NOEL or NOAEL from

Study (mg/kg/day)

Qualified Level (mg/kg/day)

Qualified Level (%) for 60 kg Human and 200 mg/day

Dose Study Reference

Tabulated Summary 2.6.7.4 Lot Number

0.14% 200 0.28 8% TX-164-2001

0.35% 450 1.58 47% TX-162-2001

0.07% 450 0.32 9% TOX153

0.88% 450 3.96 119% TX-162-2001

7.51% 200 15.0 50% TX-164-2001

GS-9237 ( , 5.96% 200 11.92 50% TX-164-2001

GS-492127 ( , ) 1.64% 200 3.28 50% TX-164-2001

0.07% 450 0.32 9% TX-162-2001

不純物FE*

不純物FC*

不純物FA*

不純物FD*

不純物FF*

不純物FB*

不純物FF''*


不純物FF'''*



2.6.6.8.7.2. Residual Solvents in Emtricitabine

Of the potential solvents used in the synthesis of FTC, only 3 are observed in the drug substance: or and (Table 30). All 3 solvents are listed in ICH Q3C and all have limits established that are below the level allowed by ICH. As such, further discussion of the residual solvents is not required.

Table 30. Qualified Levels of Residual Solvents and Proposed Limits for Emtricitabine

Solvent ICH

Classification ICH Limit (mg/day)

ICH Limita

(%)Proposed Limit

(%)

Class 2 30.0 15% 0.20%

Class 3 50 25% 1.00%

Class 3 50 25% 0.20%

a The PDE (mg/day) expressed as a percentage of the API where 200 mg/day will be dosed.

2.6.6.8.7.3. Rilpivirine: Qualification of Impurities According to ICH Q3A

, , (Figure 2) and are drug substance impurities specified at a level equal to or exceeding 0.15% were qualified according to ICH Q3A.

They were tested by spiking at 4% of the drug substance, a fair multiple of the specified level of % for and % for (see Module 3.2.S.4.1, Specification [Rilpivirine]). Neither nor spiked to TMC278 at 4% were genotoxic in the Ames test or in the mouse lymphoma assay (see Section 2.6.6.8.6). The third impurity,

, is the -isomer of TMC278. This isomer was present in all drug substance batches involved in pivotal nonclinical studies at the level of minimally 0.61%. In view of the close structure relationship with TMC278, the overage between the lowest nonclinical dose (5 mg/kg/day), and the clinical dose of 25 mg once daily, separate qualification of is not considered relevant.

2.6.6.8.7.4. Rilpivirine: Impurities with a Genotoxic Alert

is the HCl salt of (Figure 3), a starting material in an earlier synthesis route of TMC278 drug substance (see Module 3.2.S.2.6, Manufacturing Process Development [Restricted Part of Janssen Pharmaceutica’s Active Substance Master File]). On the basis of the presence of and moieties in

, its genotoxic potential was evaluated in 3 independent repeats of a bacterial reverse mutation assay (Ames) and an in vitro mammalian chromosomal aberration assay (see Section 2.6.6.8.6). The possible impact on human safety of the presence of in drug substance and drug product is evaluated in the Nonclinical Overview (Module 2.4).

不純物B* 不純物C* 不純物A*

不純物D'* 不純物D*



不純物A*

不純物A*

不純物D*

不純物D'*




Traces of and (Figure 4) may be present in TMC278 drug substance. These compounds having an moiety, but no share structural similarities with . Their genotoxic potential was evaluated in a bacterial reverse mutation test (Ames). It was concluded that neither and nor showed mutagenic potential in these studies (see Section 2.6.6.8.6).

2.6.6.8.7.5. Tenofovir DF: Qualification of Impurities in Tenofovir Disoproxil Fumarate

Process Related Impurities and Degradation Products

Over 17 impurities and degradation products related to TDF have been identified in batches of the active pharmaceutical ingredient (API) or drug product. The impurity profiles for batches of API or drug product used in nonclinical toxicology studies are provided in Tabulated Summary 2.6.7.4. In addition, another impurity ) present in clinical batches was identified after the clinical and nonclinical registration studies were completed. The major impurities are discussed below.

Two degradation products, TFV and , are metabolites of tenofovir disoproxil ( ). Tenofovir is the active moiety of the prodrug tenofovir disoproxil. PMPA is released in vivo following hydrolysis of the prodrug and has been studied extensively by subcutaneous, oral, and intravenous routes of administration in efficacy and toxicity studies described above. is a metabolite which is produced in vivo following partial cleavage of the prodrug. This compound has been shown to have cellular uptake similar to that of PMPA and, therefore, probably has poor oral bioavailability.

and are synthetic by-products and degradation products. is structurally most closely related to and, as such, is unlikely to have significant bioavailability.

The impurities ( ), , , , and have not been studied individually in vivo but,

because of their close chemical structural similarity to tenofovir disoproxil, it is anticipated that they may be orally bioavailable. If bioavailable, it is expected that they would be metabolized to release PMPA.

, and are impurities closely related to TDF. These compounds are likely to be absorbed from the intestinal tract. As they are cleaved and absorbed or absorbed and cleaved; it is believed that they would be metabolized to release PMPA. A further degradation product has also been observed, and like the , if adsorbed would likely be metabolized to PMPA.

All of these impurities have been present in material tested in toxicology studies, as well as in clinical studies. It is important to test impurities that might have limited oral bioavailability or might have local irritant properties at relatively high doses via gavage to evaluate potential GI toxicity in addition to systemic organ toxicity that they might produce. Several studies in

不純物D'*

不純物E* 不純物F*

不純物TA*

不純物TB*

不純物TC*

不純物TD* 不純物TE*

不純物TF* 不純物TG* 不純物TH*

不純物TI* 不純物TJ*

不純物TK* 不純物TL* 不純物TM*

不純物TA*


不純物TB*

不純物TB*不純物TD*

不純物TF*




rats demonstrated GI toxicity at high doses of TDF. The effects were consistently identified and were dose related. The exact components of the TDF which produced the GI toxicity were not identified. A NOEL of 30 mg/kg/day was determined for GI toxicity in rats in a 13/42 week subchronic/chronic study and a NOEL of 300 mg/kg/day was determined for the 14-day toxicity study of degraded TDF tablets (described below). A summary of the qualified level for the impurities observed at levels over 0.10% is provided in Table 31. The qualified level is calculated as:

DoseHumanWeight Humanx

100NOELx LevelObserved (%) Qualified

The observed level and the NOEL or NOAEL are listed in the table. The weight used for this calculation was 60 kg and the dose is 300 mg of TDF per day.



Table 31. Qualified Levels for Impurities of Tenofovir DF

Impurity

Maximum Observed in Toxicological Studies (%)

NOEL or NOAEL from

Study (mg/kg/day)

Qualified Level

(mg/kg/day)

Qualified Level for 60 kg Human

and with 300 mg/day Dose

Lot Number with Highest Level

Observed

Tox Study Where the Highest Observed Level was QualifiedTabulated Summary 2.6.7.4

0.22 300 0.66 13% R2000081

1.84 300 metabolite metabolite R2000081

0.26 30 0.08 2% 97-TOX-4331-002

0.28 300 0.84 17% R2000081

0.31 300 0.93 19% R2000081

0.22 300 0.66 13% R2000081

0.21 150 0.11 6% ( )

97-TOX-4331-004

0.14 300 0.42 8% R2000081

0.38 300 1.14 23% R2000081

0.13 300 0.39 8% R2000081

0.65 300 1.95 39% R2000081

0.57 30 0.17 3% 97-TOX-4331-002

0.36 300 1.08 22% R2000081

不純物TD*

不純物TB*

不純物TN*

不純物TM*

不純物TF*

不純物TG*

不純物TH*

不純物TE*

不純物TI*

不純物TJ*

不純物TL*

不純物TO*

不純物TK*




Organic Volatile Impurities

Five organic volatile impurities (OVI) are potentially present in TDF: , , ( ), , and

( or ). The ICH classification and allowed levels are summarized in Table 32. , , and are residual solvents which are present at levels lower that those specified by the ICH Q3C document “Impurities: Guideline for Residual Solvents.” and are Class 3 solvents with established safety profiles and allowed exposure at up to 50 mg/day without justification. is a Class 2 solvent with a potential maximum daily exposure of 3.0 mg from a 300 mg/day dose of TDF. This is within the "permitted daily exposure" (PDE) of 5.3 mg/day specified for this solvent in ICH Q3C(M). Therefore, these 3 solvents need no further discussion.

Table 32. ICH Classification of Organic Volatile Impurities for Tenofovir DF Drug Substance

Impurity ICH

Classification ICH Limit

(PDE, mg/day) Proposed Maximum Exposure (mg/day)

( ) Class 2 5.3 3.0

Class 3 50 0.6

Class 3 50 0.3

Not Classed none 0.3

( ) Not Classed none 0.9

a ICH limits are based on total daily dose of drug products. The drug product contains 300 mg of the tenofovir DF drug substance.

The residual solvent ( ) is mentioned in ICH Q3C as a solvent for which no is available. This impurity has not been tested as a component of the TDF toxicology

program since the GLP batches used did not contain detectable levels. However, a significant amount of toxicology literature is available for . and its oxidation products,

and , are not genotoxic {2619}. It has been tested in a developmental toxicity assay in rats in which the animals were exposed by aerosol for 6 hours/day, 5 days/week for 90 days. The compound was not teratogenic at exposure levels up to 7,100 ppm. was described as having a low order of toxicity, with a NOEL of 480 ppm exposure level. It has been tested by oral gavage in rabbits and rats {2615}. The minimum lethal dose in rabbits was 5 to 6.5 g/kg and the LD50 in rats was reported to be 11.4 g/kg {2618}, confirming the low order of toxicity. From the oral data in rats, using a 1000-fold safety factor based on the minimal toxic dose, the suggested maximum permissible oral dose in man is 0.5 mL/60 kg (0.36 mg/60 kg). A limit of 0.10% (permitting a maximum exposure of 0.3 mg/day) is specified for TDF.



, which is present in the API, has been tested in a 28-day rat study and 2 mutagenicity assays (Ames and mouse lymphoma [L5178Y TK+/- cell] assays; 1432/022; 1432/023). was shown to be mutagenic in both assays. In the 28-day oral dosing study in rats, it was shown to have a low order of toxicity. In this study, rats were dosed daily with 0, 15, 150, or 1000 mg/kg/day. The animals in the highest dose group had reduced food consumption and decreased body weights, but there was no mortality associated with any dose. The only treatment-related lesions observed at the termination of the study were inflammation, ulceration, and acanthosis/hyperkeratosis of the forestomach in the 150- and 1000-mg/kg/day dose group animals. These are lesions that would be expected following repeated dosing with a topically irritant organic compound. The NOEL in this study was 15 mg/kg/day (1432/021). The anticipated human exposure following a 300 mg daily dose of TDF containing 0.30% (the specified limit) is 0.015 mg/kg/day. The 15 mg/kg/day NOEL in the rat represents a 1000-fold safety margin for toxicity at this level of exposure.

was present in all of the TDF batches tested in the in vivo toxicology program with the highest levels (0.58%) in the initial batch ( ) used in a 13/42 week rat study. The 13-week MEL in this study was 100 mg/kg/day (Tabulated Summary 2.6.7.7.L, 97-TOX-4331-002). The rats that received a dose of 100 mg/kg/day of TDF were exposed to 0.58 mg/kg/day of . This dose is approximately 39-fold higher than the human exposure following a 300 mg dose containing 0.30% . Some groups of animals that received this relatively high exposure to for 3 months were maintained on study with continued exposure to lower levels of for a total exposure duration of 10 months. There were no unusual toxicity findings or evidence of proliferative tissue changes in any animals following this chronic exposure. Although the toxicology of has not been studied extensively, the 1000-fold margin of subchronic toxicity in rats treated with the neat compound and the extended duration of exposure to the compound in the 13/42-week study is considered adequate to qualify the proposed 0.30% with an acceptable safety margin for human exposure.

A 14-day study was conducted in rats to determine if there were unexpected toxicologic effects of degraded products of TDF tablets which had been degraded under accelerated conditions (Tabulated Summary 2.6.7.17.C, R2000081). No treatment-related findings were observed following 14 days of oral gavage dosing of either TDF or degraded TDF tablets at doses up to 300 mg/kg/day to rats. No differences between treatment with either TDF or degraded TDF tablets were noted.

Based on their impurity profiles, the multiple GLP batches of TDF tested in the toxicology program are considered, in composite, to be representative of the GMP material and support the specified limits of impurities proposed for commercial production.

2.6.6.8.7.6. Qualification of Degradation Products in Emtricitabine/Tenofovir DF Tablets

Two new degradation products not present in the drug substances have been observed in tablets placed on accelerated stability at high temperature. The trivial name for these



degradation products are /FTC Adduct (Adduct 1) and /FTC Adduct (Adduct 2). Both of these adducts form when , formed by

the hydrolysis of TDF ), reacts with one molecule of each TDF and FTC to form an adduct. The qualification of these adducts is summarized in Table 33.

Table 33. Qualification of Impurities from TDF/FTC Tablets

Impurity Max

imum

Obs

erve

d in

Tox

icol

ogic

al

Stud

ies (

%)

NO

EL o

r N

OA

EL

from

Stu

dy

(mg/

kg/d

ay)

Qua

lifie

d L

evel

(m

g/kg

/day

)

Qua

lifie

d L

evel

for

60

kg H

uman

and

300

m

g/da

y do

se

Toxicology Study Number or Reference Lot Number

-FTC adduct (Adduct 1)

0.18 300 0.54 11% TX-164-2001

-FTC adduct (Adduct 2)

0.04 300 0.12 2% TX-164-2001

The impurities and degradation products in the 2 active ingredients, FTC and TDF, as well as the tableted drug product have been identified and qualified in toxicology studies. The limits proposed for these impurities and degradation products provide an adequate safety margin when compared to the limits qualified in the individual toxicology studies.

2.6.6.8.7.7. Qualification of Degradation Products in Emtricitabine/Rilpivirine/Tenofovir DF Tablets

The impurities and degradation products present in FTC, RPV, TDF, and FTC/TDF tablets have been qualified through toxicology studies which employed drug substance from normal productions batches, laboratory scale batches with enhanced levels of impurities, and samples subjected to forced degradation conditions (high heat and humidity). There are no unique impurities or degradation products present in the FTC/RPV/TDF tablets that require qualification (Module 3.2.P.5.5, Characterization of Impurities [FTC/RPV/TDF Tablets] and Module 3.2.P.5.6, Justification of Specifications [FTC/RPV/TDF Tablets]).

不純物TB* 不純物TC*

不純物TC*

不純物TB*

不純物TC*




2.6.6.8.8. Other Studies

2.6.6.8.8.1. Tenofovir: Mutagenicity Test with GS-1278 (PMPA) Lot# in the Salmonella-Escherichia Coli/Mammalian-Microsome Reverse Mutation Assay

Tenofovir was tested at 4610, 3070, 922, 615, 307, and 92.2 g per plate in both the presence and absence of S9 mix on Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537, and Escherichia coli tester strain WP2uvrA (Tabulated Summary 2.6.7.17.C, 95-TOX-1278-006). The doses tested in an additional trial were 5000, 3330, 1000, 667, 333, and 100 g per plate with tester strains TAl00 and TA1535 only in the presence of S9 mix.

Tenofovir caused positive (6.2- and 6.8-fold) increases in the mean number of revertants per plate with tester strain TA1535 only in the presence of S9 mix. No other increase in the mean number of revertants per plate was observed with any of the remaining tester strain/activation condition combinations either in the presence or absence S9. Of note, rat liver S9 used in this study contained both microsomal and cytosolic enzymes; cytosolic phase 2 enzymes can also be induced by Aroclor and some (e.g., GSH transferases) have been shown to be involved in xenobiotic metabolic activation.

2.6.6.8.8.2. Tenofovir: Mutagenicity Test on GS-1278 (PMPA) in the L5178Y TK Mouse Lymphoma Forward Mutation Assay

The ability of TFV to induce forward mutations at the TK locus in the mouse lymphoma L5178Y cell line was evaluated (Tabulated Summary 2.6.7.17.C, 95-TOX-1278-007). Tenofovir was soluble in water at 50.0 mg/mL.

In the preliminary assay, the test article was shown to be nontoxic with and without S9 metabolic activation at concentrations of 9.77 to 5000 g/mL. In the main assay, TFV was negative at concentrations of 156 to 5000 g/mL without metabolic activation, and in the presence of S9 metabolic activation the 5000 g/mL treatment induced a small (approximately 2.5-fold) increase in the mutant frequency that exceeded the minimum criterion. Since only a single small increase was observed, TFV was considered equivocal with activation in this trial. The test article, TFV, was therefore considered negative without activation and equivocal with activation at the TK locus in L5178Y mouse lymphoma cells under the conditions used in this study.



Toxicological Findings of Tenofovir (PMPA) in SIV Efficacy Models

2.6.6.8.8.3. Tenofovir: Administration of 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) to gravid and infant rhesus macaques (Macaca mulatta): safety and efficacy studies. J Acquir Immune Defic Syndr Hum Retrovirol., 1999 Apr 1;20 (4):323-33

Tenofovir (9-[2-(R)-(phosphonomethoxy)propyl]adenine, PMPA) significantly inhibits viral reverse transcription and has been reported to sustain low virus load in SIV-infected rhesus monkeys (Tabulated Summary 2.6.7.17.C, {1787}). Based on these findings, studies were conducted to assess the safety, efficacy, and placental transfer of TFV when administered once daily subcutaneously to gravid rhesus monkeys during the second and third trimesters and their offspring (30 mg/kg/day). Fetuses (SIV-infected, N = 6; noninfected; N = 6) were monitored sonographically and maternal/fetal blood samples were collected at select time points for hematologic, clinical chemical, virologic, immunologic, and pharmacologic assessments. Newborns were delivered by cesarean section at term and nursery reared for postnatal studies. Infants were administered TFV once daily beginning on Day 2 of life until 9-months postnatal age. Results of these studies have shown significant placental transport of TFV, with peak fetal levels at 1 to 3 hours postmaternal administration; a significant and sustained reduction in viral load in SIV-infected fetuses and infants; and marked improvements in outcome (e.g., survival, growth, health) in SIV-infected offspring. However, decreased infant body weights and alterations of select serum biochemical parameters (e.g., decreased phosphorus levels, elevated ALP) have been shown to occur in 67% of TFV-treated infants, with severe growth restriction and bone-related toxicity in approximately 25% of animals studied. These data suggest that there is the potential for bone-related toxicity at chronic, high dosages, particularly in infants.

2.6.6.8.8.4. Tenofovir: The Effects of 9-[2-(R)-(phosphonomethoxy)propyl]adenine(PMPA) Treatment on Cortical Bone Strength in Rhesus Monkeys (Macaca mulatta)

The objective of this study was to evaluate 62 bones from 43 rhesus monkeys (Tabulated Summary 2.6.7.17.C, T1278-00030). The bones were derived from fetuses (4 exposed to TFV in utero during the second and third trimesters, 1 control) and SIV-infected and SIV-noninfected juveniles (22 TFV-treated, 22 untreated) following exposure to subchronic to chronic daily administration of TFV by subcutaneous injection at a dosage of 30 mg/kg/day initiated as infants or to gravid monkeys. Bones from several animals in which TFV treatment had been discontinued for several months or the dosage reduced to 10 mg/kg/day were also examined. The evaluation included assessment of bone strength, mineral content, and histomorphometric analysis. Fetal bones were not adversely affected as assessed by any of the parameters. One-half of the monkeys treated with 30 mg/kg/day for at least 4 months had increased osteoid seam widths consistent with osteomalacia. Bones from monkeys that had treatment discontinued or dose-reduction to 10 mg/kg/day were considered normal, suggesting a potential reversal of the bone changes.



Other Toxicological Findings of Tenofovir (PMPA) in SIV Efficacy Studies

Investigators from the California Regional Primate Research Center and the Tulane Regional Primate Research Center have reported results of completed and ongoing antiviral efficacy studies of TFV in SIV-infected and non-infected rhesus monkeys. Tenofovir administered subcutaneously at a daily dose of 30 mg/kg/day demonstrated potent and prolonged antiviral activity. Tenofovir administered to newborn or infant rhesus monkeys at doses of 4 to 30 mg/kg/day did not cause adverse effects in short term studies (up to 12 weeks). However, prolonged TFV treatment (generally more than 4 months of daily treatment at 30 mg/kg/day, subcutaneous) resulted in a Fanconi-like syndrome with glucosuria, aminoaciduria, hypophosphatemia, growth restriction, and bone pathology (osteomalacia; Tabulated Summary 2.6.7.17.C, {7311}). The steady state exposure (AUCss) in rhesus monkeys at this dosage is more than 30-fold that in humans following the oral administration of 300 mg/day of TDF. Clinical, biochemical, and radiographic resolution/improvement occurred with dose reduction (from 30 to 10 mg/kg/day) or discontinuation of treatment.

In another study, TFV (30 mg/kg/day, subcutaneous) treatment for 364 days reduced serum phosphorus levels (48%) and increased mean ALP activity (650%); serum calcium levels remained within normal range (Tabulated Summary 2.6.7.17.C, T1278-00034). Bone evaluations showed 1 of 5 monkeys treated with TFV had decreased bone density (L3–L5) and bone displacement (T4–T5) in the spine. The other 4 animals in this group also had some bone loss in the spine or pelvis.

Three animals (1 SIV–infected) were dosed chronically, beginning as neonates, with 10 mg/kg/day of TFV administered subcutaneously. After more than 5 years of treatment, there are no clinical, radiographic, or DXA scan (Tabulated Summary 2.6.7.17.C, {7311}) findings of an adverse effect on bone. The AUCss associated with this dosage are 3- to 18-fold greater than the human AUCss following a 300 mg/day dose of TDF.

2.6.6.9. Discussion and Conclusions

Comprehensive programs of nonclinical studies with FTC, RPV, and TDF have been conducted. The nonclinical data indicates administration of the FTC/RPV/TDF FDC tablet is unlikely to exacerbate known toxicities of the individual agents.

2.6.6.9.1. Target Organ Toxicity: Emtricitabine and Tenofovir DF

Administration of the FTC/TDF combination is unlikely to exacerbate known toxicities of individual agents. This was confirmed by the absence of any new or more marked toxicities in a 14-day rat toxicology study of the FTC/TDF tablet in comparison with studies of a similar duration with the individual agents and a 4-week toxicity study in dogs with the combination (Tabulated Summary 2.6.7.7.M and 2.6.7.7.T, TX-164-2001 and TX-164-2004, respectively). In addition, coadministration of FTC and TDF to SIV-infected pig-tailed monkeys at dose levels many times higher than the intended clinical dosages for up to 6 months had no obviously deleterious effects on the animals (Tabulated Summary 2.6.3.1.4, {5477}).



The 2 drugs exhibit different patterns of target organ toxicity. Specifically, the only significant effect of FTC identified at dose levels constituting large clinical multiples was a minor anemia. In contrast, extensive nonclinical investigations of the toxicity of TDF have shown that the bone marrow is not a target for this agent, and that the target organs for TDF are distinctly different (GI, bone, and kidney). While the toxicity profile for TDF is more diverse than that of FTC, the ample nonclinical safety database on both drugs strongly indicates that no potential for drug interaction exists and further toxicological investigations are unlikely to yield new data relevant to humans.

In a variety of in vitro and in vivo studies, FTC and TDF have shown a low potential for mitochondrial toxicity. In vitro studies to examine the potential mitochondrial effects of the combination of TDF and FTC showed no significant findings.

Further studies of longer duration with the FTC/TDF combination seem unwarranted given the lack of effects in toxicity studies of up to 4 weeks, the lack of overlapping toxicities, and the extensive clinical experience with the individual agents and the combination product, Truvada, within antiviral combination therapy for the treatment of HIV-1 infection.

2.6.6.9.2. Target Organ Toxicity: Rilpivirine

The single dose toxicity of RPV was low. The repeat-dose toxicity studies identified a number of targets. The majority of the effects induced in the highest dose tested were reversible. The increase in APTT and PT in male rats and the follicular hypertrophy and incidence of small follicles in thyroid gland of male and female rats showed incomplete recovery after a 1-month postdosing period. In dogs, microscopical lesions in liver and increased serum concentration of ALP were not completely reversed. The targets identified are described below.

Liver

Liver effects in rats and mice differed qualitatively from those in dogs. In the rodent species, predominantly hepatocellular hypertrophy occurred accompanied by effects on liver-associated serum parameters. Liver enzyme activities in serum, ALT and ALP, were more consistently increased in the mouse studies than in the studies in rats. Moreover, serum total protein concentrations, mainly due to serum albumin, were increased. However, serum triglycerides and total bilirubin (rats only) concentrations were reduced.

Mice showed some additional or different effects compared to rats. It cannot be excluded that these differences are caused by the fact that in mice much higher exposures were achieved than in rats. The hepatocellular hypertrophy in mice was accompanied by an increase in vacuolization and single cell necrosis. Electron microscopy indicated that this hypertrophy was associated with peroxisome proliferation. By contrast, the incidence of these additional effects was lower in rats. In addition, mice showed pigmentation and proliferation of Kupffer cells and increased serum concentrations of cholesterol, calcium, and inorganic phosphorus. The interrelationship of the serum chemistry effects is doubtful and an association with the Kupffer cell effects is not very likely.



The increased serum urea concentration of female mice in the high dose group dosed with 320 mg/kg/day is in line with the increased food intake in that group.

In dogs, the liver effects changed with the duration of treatment. In the 1-month study, perivascular inflammatory reactions together with multifocal perivascular fibrosis and single cell necrosis were noted in the central part of the lobules. Moreover, increased MPS aggregates occurred and multifocal bile duct proliferation. These effects seem to be associated with increased serum concentrations of cholesterol and total bilirubin and increased activities of ALP and ALT. In the 6-month study, pigment-laden perivascular macrophages were noted; and in males only, prominent brown pigment in the gall bladder epithelium. The pigment in the macrophages was not stainable with Perl’s stain, indicating that it probably was of lipogenic origin. This pigmentation was the only liver effect observed after 3 months of dosing (in high dose males only), indicating the relatively slow development of this effect, which also applies for the pigmentation in the gall bladder. In the 12-month dog study, pigmentation of hepatocytes and canaliculi was noted together with prominent brown pigment in gall bladder epithelium, both in males and females. The histopathological effects were associated with an increased serum concentration of total bilirubin and with an increase in serum ALP activity.

In the studied species, liver effects did not occur at the lowest dose tested.

Thyroid gland

Effects on the thyroid gland in rats were characterized by increased organ weight, hypertrophy of follicular epithelium, and reduced serum concentrations of T4. The effects occurred similarly in males and females and were noticed after 7 days of treatment, even at the low dose of 40 mg/kg/day. Upon longer duration of treatment, increased serum concentrations of TSH were recorded and also a differential effect on T3. The serum concentrations of this thyroid hormone (T3) were less reduced than those of T4, and in some studies were not affected or even increased. The effects on thyroid gland are thought to be caused by an increased clearance of T4. A well-known and frequent cause of the enhanced T4 clearance in rats is induction of T4-uridine diphosphate glucuronosyltransferase (UDPGT) in liver. Ex vivo enzyme induction determinations in liver homogenate from rats treated with RPV for 6 months showed only a 20% higher UDPGT activity of the high-dose group dosed with 400 mg/kg/day compared to controls. There are no indications that RPV has a direct effect on thyroid gland or a particular affinity to thyroidal tissues. The thyroid gland effects occurred in all studies at the low dose and therefore prevented establishment of a NOAEL in rats.

The minimal follicular cell hypertrophy that was noted in thyroid gland of immature female cynomolgus monkeys in the 8-week study is probably associated with the young age of these animals at the start of the study. However, an effect by RPV cannot be excluded in view of the higher incidence of the effect in the groups treated with the test article compared to the control group. In this study, no levels of thyroid hormones or TSH were determined. Microscopical evaluation of other organs did not show any cause for the thyroidal effect.



Pituitary gland

Effects on the pituitary gland occurred only in rats and comprised an increase of swollen and vacuolated cells in the pars distalis. These effects are considered secondary to the thyroid gland effects. The indicated cells produce TSH. The low concentration of T4 in the circulation is the feedback signal for the pituitary gland to produce more TSH. The effects on the pituitary gland were noted in males treated with the low dose in the 6-month rat study and hence prevented establishment of a NOAEL.

Kidneys

Kidney changes were seen in dogs in the 12-month study dosed with the high dose of 40 mg/kg/day, and in the 1-month study with transgenic mice and the 3-month study with CD-1 mice, both at the high dose of 320 mg/kg/day (and in the transgenic mice also at 80 mg/kg/day). The changes in the dog comprised moderate acute interstitial nephritis in 2 high dose males and minimal to slight corticomedullar mineralization in 3 out of 4 high dose females.

In the 1-month study with transgenic mice, all females, 3 out of 10 males treated with 320 mg/kg/day, and 1 out of 10 males at 80 mg/kg/day showed minimal to moderate degenerative or necrotic nephropathy. Minimal to moderate nephropathy was noted in half the number of female mice dosed with 320 mg/kg/day in the 3-month study. In the carcinogenicity study with CD-1 mice, no increased incidence of nephropathy, mortality associated with renal effects, or renal neoplasia was noted.

For the nephritis in dogs, a test article treatment relationship cannot be excluded. However in mice, such a relationship seems to be clear. The mechanism of these kidney effects is not clear taking into account the low percentage of the dose excreted via urine (see Module 2.6.4, Pharmacokinetic Written Summary, Section 2.6.4.6.1).

Adrenal glands

In all species, except rabbits, involved in nonclinical safety studies with RPV, an effect on adrenal gland has been demonstrated. The levels of adrenal hormones or their precursors and ACTH in serum of rats and dogs, and the results of the in vitro studies with guinea pig primary cell cultures and homogenates of adrenal gland cortex of dog indicate that RPV partially inhibits cytochrome P450 isozyme 21 (CYP21) (see Figure 5). Cytochrome P450 isozyme 21 catalyzes the conversion from progesterone and 17 -hydroxyprogesterone to 11-deoxycorticosterone and 11-deoxycortisol, respectively; the direct precursors of respectively corticosterone and cortisol. A partial block of this pathway will lead to a reduced production of downstream hormones of which cortisol and corticosterone are the most important. Moreover, an accumulation of the substrates of CYP21, progesterone and 17 -hydroxyprogesterone and more upstream hormones may occur. In vivo, the reduced serum levels of cortisol and corticosterone due to inhibition of CYP21 will trigger an ACTH-response from the pituitary. The increased stimulus of the adrenal cortex by ACTH will partially counteract the reduction of the cortisol and corticosterone synthesis, but at the same time will increase the accumulation of the CYP21 substrates.



An additional effect of the CYP21 inhibition, notably in species that have a significant androgenic adrenal pathway, is that the increased ACTH stimulus and subsequent accumulation of progesterone and 17 -hydroxyprogesterone may lead to an increased androgen production. 17 -hydroxypregnenolone and 17 -hydroxyprogesterone are converted to DHEA and androstenedione, respectively; catalyzed by 17, 20 lyase (CYP17). To investigate the impact of the stimulation of the androgenic pathway, a study was conducted in cynomolgus monkeys. This monkey strain and all nonhuman primates share with man a significant androgenic adrenal pathway. The 8-week study in cynomolgus monkeys showed that RPV inhibits both CYP21 and CYP17. This dual effect led to a clear accumulation of progesterone and 17 -hydroxyprogesterone, both being substrates for both CYP21 and CYP17, and a reduction of the unchallenged serum levels of androstenedione and DHEA, and of the response of these androgens to ACTH challenge.

Figure 5. Synthetic Pathways for Adrenal Steroid Synthesis in Nonhuman Primates and Man

The first step in adrenal steroid synthesis is the combination of acetyl CoA and squalene to form cholesterol, which is then converted into pregnenolone. The enclosed area contains the core steroidogenic pathway utilized by the adrenal glands and gonads. 17 : 17 -hydroxylase (CYP17, P450c17); 17,20: 17,20 lyase (also mediated by CYP17); 3 : 3 -hydroxysteroid dehydrogenase; 21: 21-hydroxylase (CYP21A2, P450c21); 11 : 11 -hydroxylase; (CYP11B1, P450c11); 18 refers to the two-step process of aldosterone synthase (CYP11B2, P450c11as), resulting in the addition of an hydroxyl group that is then oxidized to an aldehyde group at the 18-carbon position; 17 R: 17 -reductase; 5 R: 5 -reductase; DHEA: dehydroepiandrostenedione; DHEAS: DHEA sulfate; A: aromatase (CYP19).



The effects of RPV on adrenal gland and its hormonal biosynthetic pathways are more prominent than the effects on gonads and their pathways (see below in more detail). This may be explained by the affinity of RPV for adrenal gland in repeat-dose studies in dogs and of radiolabel following the rat single-dose quantitative whole body autoradiography (QWBA) studies with [14C]RPV (see Module 2.6.4, Pharmacokinetic Written Summary, Section 2.6.4.4.2). In the QWBA studies, no particular affinity for gonads was noted.

Effects on adrenal hormones were noted in all repeat-dose dog studies with treatment duration of at least 7 days. Apparently, the effect needs some time to become manifested. This may explain why no effects were noted in the single oral dog study with a CRF challenge 24 hours after dosing. The fact that effects on adrenal pathways were already detected at the lowest dose levels in the dog and cynomolgus studies prevented a NOAEL to be established.

Ovaries

The effects in ovaries of dogs after at least 1 month of treatment with RPV were characterized by increased numbers of tertiary follicles, (cystic) luteinized follicles, and in some dogs, corpora lutea. These effects did not intensify over time after 3 months of dosing. The age of the dogs at the start of dosing in the 1-month studies was between 6.5 and 8 months. Probably, the still preadolescent age at the end of the 1-month studies has contributed to the more prominent effect in the test article-treated female dogs, compared to the controls that appeared still sexually immature, than in the longer duration studies. In the 1-month studies, the ovarian effects were associated with changes in the other parts of the female genital tract and with the prominent activation of the mammary glands. It is conceivable that the mechanism of action of the effects on the ovaries in dogs is associated with the inhibition of CYP21 and CYP17 by RPV. But the exact mechanism of action of the observed early maturation (1-month studies) or activation of ovaries in the dog could not be evaluated from the general toxicity studies. Effects of RPV at the level of serum concentrations of progesterone and estradiol were difficult to evaluate due to the estrous cycle of the dogs.

The absence of ovarian effects in rats and immature cynomolgus monkeys provided no contribution to the establishment of the mechanism of the ovarian effect in dogs. The absence of the ovarian effects in the cynomolgus monkeys cannot be explained by lack of sensitivity of that species for the endocrine effects of RPV. The compound had a clear effect on adrenal CYP21 and CYP17 in that species concluded from the increases in serum progesterone and 17 -hydroxyprogesterone and from the reduction of DHEA and androstenedione. The more early stage of development of the monkeys (approximately 20 months of age at necropsy, at least 1 year prior to the onset of puberty) compared to the dogs (approximately 7 months of age at necropsy, a few months prior to the onset of puberty) may have contributed to the difference in response. However, a dog-specific effect cannot be excluded.



Testes

The Leydig cell changes seen in dogs only were characterized by bilateral diffuse swelling or hypertrophy noted at the end of the 1 and 12 months of treatment, and hyperplasia and hypertrophy after 3 and 6 months of treatment. The effects were minimal in the 1-month study, most prominent after 3 months of treatment, and again minimal after 6 and 12 months. It cannot be excluded that the minimal Leydig cell hypertrophy is associated with the effects of RPV on CYP21 and CYP17. However, the exact mechanism of action could not be evaluated in these general toxicity studies in which there were no effects on serum testosterone or LH. The absence of similar effects in mice and rats seems to indicate that the Leydig cell hypertrophy may be a dog-specific effect.

In addition to the Leydig cell effects, marginal findings were noted in the seminiferous tubules and secondary in the epididymides. The marginally increased focal unilateral atrophic tubules seen at the high dose after 3 and 6 months of treatment was associated with marginal cellular debris in the epididymides, in the mid- and the high-dose groups. Reviewing all the data on testes after completion of the development program, this effect and the reduced spermiogenesis leading to reduced numbers of spermatozoa in the epididymides of a single animal in the mid- and high-dose groups after 6 months of treatment are not considered of toxicological relevance. The main reasons for this conclusion are the similarity with background data and the lack of effects on spermiogenesis in the 12-month study upon staging of the spermatogenic cycles.

Red blood cells

In mice, rats, and dogs, RPV caused a small (hardly ever exceeding 10%) reversible reduction of red blood parameters in combination with regenerative signs. The effects usually occurred only in the high-dose groups in each of the studies with the indicated species. There were no clinical signs or symptoms of anemia noted in any of the species. Since exposures at the high doses in the various species differed considerably, it seems that the effect on RBCs is not directly associated with exposure. Regeneration was evidenced by extramedullar hematopoiesis leading to increases in reticulocyte counts. The mechanism of action of the effect on red blood parameters could not be established in any species. However, there were no signs indicative of bone marrow suppression. The increased cellularity in bone marrow of female mice at the high dose is likely due to an increase of erythroid elements compensatory to the decrease of RBCs in the circulation. The increased myeloid/erythroid ratio in bone marrow of male mice is assumed due to an increase in myeloid precursor cells compensating the decrease of lymphocytes in the circulation. Moreover, in the mouse and rat carcinogenicity studies no effects were noted on bone marrow and hemopoietic cells.



Coagulation

In male rats, increased values for APTT and PT were noted in the 2-week pilot study with RPV at doses of 1500 mg/kg/day and higher, and in the 6-month study at all doses of RPV from 40 up to 400 mg/kg/day. The effects were not observed in the 1-month study with RPV at doses up to 160 mg/kg/day. The cause of this discrepancy is not known. However, it cannot be excluded that the development of the effect at lower doses takes more time than at higher doses. In other rat studies, APTT and PT were not determined. Increased values of intrinsic (APTT) and extrinsic (PT) coagulation times indicate a reduced efficacy of these major coagulation pathways. The relevance of this limited effect (less than a 30% increase) that was not associated with any bleeding, did not demonstrate a dose effect relationship, and which occurred in males only is questionable. Since the effect occurred also in the low dose, it contributes to the factors preventing the establishment of a NOAEL in rats.

2.6.6.9.3. Target Organ Toxicity: Emtricitabine/Rilpivirine/Tenofovir DF

The toxicity profiles of the 3 agents differ substantially. The only toxicity observed in chronic animal studies with FTC was mild, reversible anemia in mice and minor decreases in erythrocyte counts/increases in MCH in monkeys at large multiples of clinical exposure (168-fold in mice; 26-fold in monkeys); therefore, these hematological findings are not considered relevant to clinical use. The principal target organs of toxicity following administration of RPV were the adrenal glands and associated steroid biosynthesis (mouse, rat, dog, and cynomolgus monkey), liver (mouse, rat, and dog), thyroid and pituitary glands (rat), kidney (mouse, dog), male and female reproductive organs (dog), the hematopoietic system (mouse, rat, and dog), and the coagulation system (rat). In general, effects were reversible. Effects on the liver, kidney, hematopoietic system, and coagulation system were mild and occurred at higher doses and exposures. The principal target organs of toxicity following oral administration of TDF were the kidney (karyomegaly, tubular degeneration), bone, and GI tract (in rodents). Given that kidney effects (nephropathy) with RPV have been observed in mice and dogs at high doses, and that the routes of excretion differ for RPV and TDF, renal toxicity is not anticipated to be an issue with the combination product. Given that pharmacokinetic interactions are unlikely based on nonclinical (Module 2.6.4) and clinical (Module 2.7.2) data, and that the target organ profiles are different, administration of the combination product is unlikely to exacerbate known toxicities of the individual agents.

2.6.6.9.4. Genetic Toxicology: Emtricitabine and Tenofovir DF

Only TDF had positive findings in genotoxicity studies (mouse lymphoma cell assay and UDS assay). There was no evidence of exacerbation of mutagenic potential in in vitro genotoxicity tests (Ames test and mouse lymphoma assay) with the combination of FTC and TDF as compared to the agents alone.



2.6.6.9.5. Genetic Toxicology: Rilpivirine

The potential of RPV to induce mutagenesis has been investigated in vitro in the Ames test and the mouse lymphoma assay, and in vivo in the mouse bone marrow micronucleus test. In the test systems, RPV as base or as the HCl salt was not able to induce mutations or chromosome damage at the highest feasible concentration or dose tested. The following is considered relevant for the interpretation of the in vitro genetic toxicity data.

Metabolism studies indicated that rat S9 mix used as metabolism enhancer in the Ames test and mouse lymphoma assay had a poor capacity to generate metabolites M30 and M31. These metabolites occur in man and are the result of a Michael addition to the

moiety of RPV. The metabolic pathway to M30 and M31 encompasses at least a reactive epoxide. Due to the poor capacity of rat S9 to generate M30 or M31, the reactive intermediate was not present in the Ames or mouse lymphoma assay; and therefore its potential to induce genotoxic effects was not tested. Since some batches of human S9 appeared to generate at least M30, an additional Ames test was done with human S9 as metabolism enhancer. The test results were optimized by selecting a batch of human S9 that yielded M30 in the Ames test at least at the approximate level of M30 detected in feces in the human metabolism study. Rilpivirine did not induce mutations under these test conditions. Moreover, RPV did not demonstrate a clastogenic or aneuploidic effect in bone marrow of mice (Tabulated Summary 2.6.7.9.B, TMC278-Exp5538 and Addendum to TMC278-Exp 5538 [FK4259]). The concentration of M30 in mice is similar to that in man (see Module 2.6.4, Pharmacokinetic Written Summary, Section 2.6.4.5). So the metabolic pathway with the potentially reactive intermediates was involved in this in vivo genotoxicity study.

The structures of metabolites M30 and M31 do not comprise any alerts for genotoxicity. In view of the absence of structural alerts and the absence of indications for genotoxicity in the Ames test with human S9 and the bone marrow micronucleus study in mice, neat genotoxicity testing of M30 and M31 was not considered necessary.

The moiety of RPV generates a mutagenic alert. The drug substance impurities, , , and , contain the same moiety and were for that reason evaluated for their mutagenic potential. Only , tested as its HCl salt , increased the mutation frequency in the Ames test. contains also a

, whereas the others do not. Therefore, it is concluded that the mutagenic potential of the moiety is low and that the mutagenicity of is due to its

moiety.

The probability that genotoxic impurity could contribute significantly to an increased incidence of tumors in man upon administration of RPV is discussed in detail in the Nonclinical Overview (Module 2.4).

不純物D* 不純物E* 不純物F*不純物D’*不純物D*

不純物D*

不純物D*

不純物D*




2.6.6.9.6. Genetic Toxicology: Emtricitabine/Rilpivirine/Tenofovir DF

Of the 3 compounds, only TDF had positive findings in genotoxicity studies. There was no evidence of increased mutagenic potential with the combination of FTC and TDF. Studies with the 3 drug combination FTC/RPV/TDF are not considered likely to alter the genotoxicity profile compared to the agents alone.

2.6.6.9.7. Carcinogenicity: Emtricitabine and Tenofovir DF

TDF and FTC have both demonstrated low carcinogenic potential in conventional 2-year bioassays at exposures that exceeded (TDF) or far exceeded (FTC) human exposures at the therapeutic dose. It is considered unlikely that combination dosing would change this profile, as no exposure difference would be expected and no exacerbation of toxicity is expected.

2.6.6.9.8. Carcinogenicity: Rilpivirine

The carcinogenic potential of RPV has been studied with the HCl salt by means of 2-year carcinogenicity studies in rats and in mice.

The exposures to RPV in the mouse study were as could be expected on the basis of the previous studies in that species. However, in the rat carcinogenicity, the exposure on Day 1 of the study was lower than was anticipated on the basis of the results of the bridging study with the base and the HCl salt. The toxicokinetic data generated after 6 months of dosing in the rat study showed that exposure had declined during the study. In consultation with the US Food and Drug Administration (FDA) it was decided to continue the study and to keep the satellite animals for an additional sampling after 9 months of dosing. That sampling showed that exposure after 9 months of dosing was not different from that after 6 months of dosing. The outcome of the study in terms of a dose-related increase in neoplastic lesions illustrates that the study objectives were met. For that reason, the study is considered valid.

Hepatocellular adenomas and carcinomas are common spontaneous liver neoplasms in rodents {10217}. In general, hepatocarcinogens are divided into genotoxic and nongenotoxic agents {15694}. Since RPV is not genotoxic in a battery of in vitro and in vivo assays, the neoplastic lesions in liver observed in the mouse and rat carcinogenicity studies are considered a consequence of a nongenotoxic mechanism of action of RPV, rather than an expression of a direct carcinogenic potential of the compound. For nongenotoxic carcinogens, several mechanisms of action have been reported for rodent liver neoplasm development, including phenobarbital-like cytochrome P450 induction {15695}. The increased liver weight, associated with signs of enzyme induction, caused by RPV was already evident in the repeat dose general toxicity studies. Ex vivo hepatic enzyme activity evaluated in the 3-month mouse study and the 6-month rat study showed that RPV caused strong induction of the CYP4A family in male and female mice and male rats; and induction of the CYP3A family in female rats (Module 2.6.4, Section 2.6.4.5.1.2). Electron microscopy of mouse liver noted peroxisome proliferation, a lesion commonly associated with CYP4A induction {15696}.This pattern of liver enzyme induction correlates well with the incidence of hepatocellular adenomas and carcinomas {15696}.



The repeat-dose toxicity studies in rats have demonstrated, without exception, effects on the thyroid gland often associated with effects in the pituitary gland, and on the thyroid hormones and TSH. All data point towards a mechanism by which RPV causes an increased clearance of thyroid hormones and a subsequently activated feedback on the thyroid gland by TSH synthesized in the pars distalis of the pituitary gland, rather than having a direct effect on thyroid hormone synthesis. Furthermore in rat liver, RPV caused induction of UDPGT, the most important enzyme involved in the metabolism and clearance of thyroid hormones. UDPGT induction is a well-known cause of increased thyroid hormone clearance and upon life-long occurrence associated with the development of follicular adenomas and carcinomas {15703}.

No neoplastic lesions were observed in adrenal glands in spite of the high affinity of RPV for this organ.

Taking into account the results of the genotoxicity and the carcinogenicity studies, it is concluded that RPV has not shown any potential to induce direct DNA-related effects. The relevance for man of the epigenetic carcinogenic effects on mouse and rat liver and rat thyroid gland is discussed in the Nonclinical Overview (Module 2.4).

2.6.6.9.9. Carcinogenicity: Emtricitabine/Rilpivirine/Tenofovir DF

Tenofovir DF and FTC have both demonstrated low carcinogenic potential in conventional 2-year bioassays. For RPV, the relevance for man of the epigenetic carcinogenic effects in liver and thyroid is questionable. It is considered unlikely that combination dosing would change this profile, as no exposure difference would be expected and no exacerbation of toxicity is expected. Given the difficulty of extrapolating rodent results to humans and the large number of animals required to carry out these studies, the conduct of carcinogenicity studies with the FTC/RPV/TDF combination is not justifiable.



2.6.6.9.10. Reproductive Toxicity: Emtricitabine and Tenofovir DF

In terms of reproductive toxicity, the reproductive and developmental NOELs for the individual agents were at exposure levels well above human exposures. With no expected pharmacokinetic or toxicodynamic interactions between FTC and TDF, further studies with the FTC/TFV combination are not considered necessary. There were no effects on embryo-fetal development in rats or rabbits when FTC and TDF were tested individually. No cause for concern has been identified and studies with the combination are unlikely to show new effects. A delay in sexual maturation was observed in F1 generation rats after exposure to high (maternally toxic) doses of TDF and slightly longer oestrous cycles were observed in F1 generation rats after exposure to high doses of FTC. No other significant effects were noted and NOELs were at exposures above human exposures. As with other reproductive toxicity tests, a repeat of this test with the FTC/TDF combination is unlikely to add any new information.

2.6.6.9.11. Reproductive Toxicity: Rilpivirine

The male and female rat fertility studies and peri- and postnatal developmental studies were conducted after the HCl-salt was chosen for further development. In view of the minor testicular effects of TMC278 in dogs characterized by marginal to limited Leydig cell hypertrophy, a separate male rat fertility study was conducted with up to the highest feasible dose of TMC278 (1500 mg/kg/day).

The reproductive and developmental toxicity studies did not demonstrate any effects on male or female fertility, fecundity, maternal behavior after parturition and during weaning, or development of offspring from dams treated with TMC278 during pregnancy and lactation. The results of the embryo-fetal developmental studies in rats and rabbits with the highest feasible exposures to TMC278 demonstrated the absence of a potential for teratogenicity at high exposures to the rabbit dams. The exposure to the rat dams elicited effects at the level of the thyroid gland, which were also found in general toxicity studies.

In toxicity studies with juvenile rats or with dogs and cynomolgus monkeys completed prior to the onset of adolescence/adulthood, no additional targets were noted and no increased sensitivity to the effects found in adult animals was evident.

2.6.6.9.12. Reproductive Toxicity: Emtricitabine/Rilpivirine/Tenofovir DF

Emtricitabine, RPV, and TDF have not shown significant adverse effects in reproductive and developmental toxicity studies. The combination of the 3 components is not expected to have an altered reproductive toxicity profile compared with that of the individual agents.

2.6.6.9.13. Metabolites/Impurities: Emtricitabine/Tenofovir DF

There are no major metabolites of either FTC or TDF that require independent study. The identified metabolites have been assessed as part of the routine toxicology and qualification (of impurities) studies.



Expected impurities in the FTC/TDF portion of the tablet were assessed in a 14-day rat toxicology study. No new excipients are present in the FTC/TDF combination; the excipients have been tested as part of the FTC, TDF, and the FTC/TDF (Truvada) development program.

2.6.6.9.14. Metabolites/Impurities: Rilpivirine

The RPV drug substance contains 3 impurities at levels that need qualification according to ICH Q3A. Two impurities, and , were qualified in an Ames test and a mouse lymphoma assay and in a 1-month oral rat study using RPV spiked with 4% of each of the impurities. None of the impurities induced a difference from the effects caused by TMC278.

The third impurity, , is the -isomer of RPV. This isomer was present in all drug substance batches involved in pivotal nonclinical studies at the level of minimally 0.61%. In view of the close structure relationship with RPV and the overage between the lowest nonclinical dose (5 mg/kg/day) and the clinical dose of 25 mg once daily, it is concluded that

up to 7.44% of the daily dose of RPV is unlikely to incur untoward effects in man (average body weight 60 kg).

Three low level drug substance impurities, , , and , containing an moiety were evaluated for their mutagenic potential. Only ,

tested as its HCl salt , induced a mutagenic response and a clastogenic response. It is concluded that the mutagenic potential of the moiety is low and that the genotoxicity of is due to its . The genotoxic impurity

is controlled at a level well below that associated with the threshold for toxicological concern (TTC) {15703} for a daily intake of more than 12 months. The TTC is 1.5 μg/day for adults. With a daily dose of 25 mg RPV, the maximally allowable level of the impurity should be lower than 60 ppm. The actual level is well below 10 ppm.

2.6.6.9.15. Metabolites/Impurities: Emtricitabine/Rilpivirine/Tenofovir DF

There are no new impurities that require qualification in the combination FTC/RPV/TDF/product. All anticipated impurities have been qualified as part of the development programs for the individual agents, or for the Truvada development program.

2.6.6.9.16. Comparative Exposures and Safety Margins

Cross-species comparisons of exposure, relative safety margins and the relevance of these effects for human health are discussed in Module 2.4, Nonclinical Overview, Section 2.4.4.9.2.


不純物A*

不純物D*

不純物D’*


不純物A*

不純物D*

不純物D*

不純物D*




2.6.6.9.17. Conclusions

A comprehensive nonclinical pharmacology/virology, pharmacokinetic, and toxicology program was undertaken in support of the registration of FTC and TDF. The results of these evaluations were presented in detail in the original licensing application and subsequent submissions for Emtriva and Viread, respectively. A small number of key studies were conducted using the combination of FTC and TDF. Similarly for RPV, a complete nonclinical package is available, including a comprehensive package of drug disposition studies; safety pharmacology studies; single-dose oral toxicity studies in mice, rats and dogs; repeat-dose oral toxicity studies in mice (up to 3 months), rats (up to 6 months), nonpregnant rabbits (up to 5 days), dogs (up to 12 months), and immature female cynomolgus monkeys (up to 8 weeks); genotoxicity tests both in vitro and in vivo; 2-year oral carcinogenicity studies in mice and rats; and a full developmental and reproductive toxicity program. The overall program including the data from the combination and individual agent studies is considered adequate to support the safety of the FTC/RPV/TDF combination tablets based on the following considerations.

The toxicity profiles of the 3 agents differ substantially. The only toxicity observed in chronic animal studies with FTC was mild, reversible anemia in mice and minor decreases in erythrocyte counts/increases in MCH in monkeys at large multiples of clinical exposure (168-fold in mice; 26-fold in monkeys); therefore, these hematological findings are not considered relevant to clinical use. The principal target organs of toxicity following administration of RPV were the adrenal glands and associated steroid biosynthesis (mouse, rat, dog, and cynomolgus monkey), liver (mouse, rat, and dog), thyroid and pituitary glands (rat), kidney (mouse, dog), male and female reproductive organs (dog), the hematopoietic system (mouse, rat, and dog), and the coagulation system (rat). In general, effects were reversible. Effects on the liver, kidney, hematopoietic system and coagulation system were mild and occurred at higher doses and exposures. The principal target organs of toxicity following oral administration of TDF were the kidney (karyomegaly, tubular degeneration), bone, and GI tract (in rodents). Given that kidney effects with RPV have been observed in mice and dogs at high doses, and that the routes of excretion differ for RPV and TDF, renal toxicity is not anticipated to be an issue with the combination product. Given that pharmacokinetic interactions are unlikely and that the target organ profiles are different, administration of the combination product is unlikely to exacerbate known toxicities of the individual agents.

NRTIs carry a class labeling for mitochondrial toxicity; however, both FTC and TDF have shown a low potential for mitochondrial toxicity in long-term toxicity studies. The potential for mitochondrial toxicity of RPV was evaluated by in vitro assessment of the inhibitory activity on human polymerase (see Module 2.6.2). However, as mitochondrial toxicity is generally less relevant for NNRTIs than NRTIs, and as RPV is not anticipated to significantly increase the exposure of FTC or TDF, the potential for exacerbating mitochondrial toxicity is low.



Of the 3 compounds, only TDF had positive findings in genotoxicity studies (mouse lymphoma cell assay and UDS assay). The combination of FTC and TDF in a mouse lymphoma cell assay did not worsen the genotoxic potential of TDF. Tenofovir DF did not have significant effects in the long-term carcinogenicity studies. Rilpivirine does not show any potential for direct DNA-related effects, and the relevance of the epigenetic carcinogenic effects on mouse and rat liver and rat thyroid are not considered relevant for humans.

Emtricitabine, RPV, and TDF have not shown significant adverse effects in reproductive and developmental toxicity studies, and the combination of the 3 components is not expected to have an altered reproductive toxicity profile compared with that of the individual agents.

The FTC/RPV/TDF combination is not anticipated to produce any new metabolites. No new excipients or impurities are present in the FTC/RPV/TDF tablet.

The combination of FTV, RPV, and TDF is not anticipated to exacerbate known toxicities or lead to new toxicities. Emtricitabine has an established clinical safety profile with no significant toxicities observed. Potential toxicities related to RPV that were observed preclinically, but have not been observed in Phase 1 and 2 clinical studies to date, include effects on the adrenal system and associated changes in 17-OH progesterone and cortisol levels, and liver and thyroid toxicity. The known clinical toxicities for TDF are renal and bone toxicity.

The absence of nonclinical safety studies with the combination is in accordance with the CHMP Guideline on the Non-Clinical Development of Fixed Combinations of Medicinal Products (EMEA/CHMP/SWP/258498/2005, January 2008). There are no anticipated clinically relevant pharmacokinetic or toxicological interactions expected in the FTC/RPV/TDF combination. The Scientific Advice Working Party agreed with the assessment that no further nonclinical studies are needed to support the FTC/RPV/TDF FDC based on the comprehensive nonclinical data set for rilpivirine and the long-term safety from the Phase 2b trial TMC278-C204 (EMEA/CHMP/SAWP/670243/2009 corrigendum). Further, extensive clinical safety data are available for the approved drugs FTC, TDF, and the FTC/TDF FDC product Truvada. Additionally, data from the Phase 2b and Phase 3 trials with RPV and Truvada supports the use of these products (see Module 2.7). The clinical data, along with the lack of overlapping toxicity in animals, support the safety of the new combination product.



2.6.6.10. Tables and Figures

The tables and figure have been incorporated into the text.



2.6.6.11. References

1787 Tarantal A, Marthas ML, Shaw J-P, Cundy KC, Bischofberger N. Administration of 9-[2-(R) (phosphonomethoxy)propyl]adenine (PMPA) to gravid and infant rhesus macaques (Macaca mulatta): safety and efficacy studies. J Acquir Immune Defic Syndr Hum Retrovirol 1999 Apr 1;20 (4):323-33.

1790 Tennant BC, Baldwin BH, Graham LA, Ascenzi MA, Hornbuckle WE, Rowland PH, et al. Antiviral activity and toxicity of fialuridine in the woodchuck model of hepatitis B virus infection. Hepatology 1998 Jul;28 (1):179-91.

2615 Kirwin CJ, Galvin JB. Ethers. In: Clayton GD, Clayton FE, eds. Patty's Industrial Hygiene and Toxicology. New York: John Wiley & Sons, Inc.; 1993: Vol. 11, Part A, p. 464.

2618 Kimura ET, Ebert DM, Dodge PW. Acute toxicity and limits of solvent residue for sixteen organic solvents. Toxicol Appl Pharmacol 1971 Aug;19 (4):699-704.

2619 Brooks TM, Meyer AL, Hutson DH. The genetic toxicology of some hydrocarbon and oxygenated solvents. Mutagenesis 1988 May;3 (3):227-32.

5477 Shen A, Zink MC, Mankowski JL, Chadwick K, Margolick JB, Carruth LM, et al. Resting CD4+ T lymphocytes but not thymocytes provide a latent viral reservoir in a simian immunodeficiency virus-Macaca nemestrina model of human immunodeficiency virus type 1-infected patients on highly active antiretroviral therapy. J Virol 2003 Apr;77 (8):4938-49.

6120 Richardson FC, Tennant BC, Meyer DJ, Richardson KA, Mann PC, McGinty GR, et al. An evaluation of the toxicities of 2'-fluorouridine and 2'-fluorocytidine-HCl in F344 rats and woodchucks (Marmota monax). Toxicol Pathol 1999 Nov-Dec;27 (6):607-17.

6124 Lewis W, Griniuviene B, Tankersley KO, Levine ES, Montione R, Engelman L, et al. Depletion of mitochondrial DNA, destruction of mitochondria, and accumulation of lipid droplets result from fialuridine treatment in woodchucks (Marmota monax). Lab Invest 1997 Jan;76 (1):77-87.

7311 Van Rompay KKA, Brignolo LL, Meyer DJ, Jerome C, Tarara R, Spinner A, et al. Biological effects of short-term or prolonged administration of 9-[2-(phosphonomethoxy)propyl]adenine (tenofovir) to newborn and infant rhesus macaques. Antimicrob Agents Chemother 2004 May;48 (5):1469-87.

10217 Chen D, Misra A, Garg A. Clinical review 153: Lipodystrophy in human immunodeficiency virus-infected patients. J Clin Endocrinol Metab 2002;87 (11):4845-56.



12716 Hammer SM, Eron JJ, Jr., Reiss P, Schooley RT, Thompson MA, Walmsley S, et al. Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. JAMA 2008;300 (5):555-70.

14065 Gazzard BG. British HIV Association Guidelines for the treatment of HIV-1-infected adults with antiretroviral therapy 2008. HIV Med 2008;9 (8):563-608.

15207 Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human Services. December 1, 2009; 1-161. Available at http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf.

15639 Fridericia LS. Die Systolendauer im Elektrokardiogramm bei normalen Menschen und bei Herzkranken. Acta Med Scand 1920;53:489-506.

15694 Grasso P, Hinton RH. Evidence for and possible mechanisms of non-genotoxic carcinogenesis in rodent liver. Mutat Res 1991;248 (2):271-90.

15695 Holsapple MP, Pitot HC, Cohen SM, Boobis AR, Klaunig JE, Pastoor T, et al. Mode of action in relevance of rodent liver tumors to human cancer risk. Toxicol Sci 2006;89 (1):51-6.

15696 Lake BG. Species differences in the hepatic effects of inducers of CYP2B and CYP4A subfamily forms: relationship to rodent liver tumour formation. Xenobiotica 2009;39 (8):582-96.

15699 van Bladeren PJ, Delbressine LP, Hoogeterp JJ, Beaumont AH, Breimer DD, Seutter-Berlage F, et al. Formation of mercapturic acids from acrylonitrile, crotononitrile, and cinnamonitrile by direct conjugation and via an intermediate oxidation process. Drug Metab Dispos 1981;9 (3):246-9.

15703 Capen CC. Mechanisms of Toxicity : Thyroid Follicular Cells. Thyroid and Parathyroid Toxicology 1999:42.

15704 MacEnzie WF, Boorman GA. Pituitary gland. Hypertrophy/ hyperplasia of thyrotrophs in the pituitary gland is a well-known histological finding related to a compensatory increased TSH demand in the rat In: Pathology of the Fischer rat (Boorman G.A. et al.), Academic Press, Inc., San Diego, 1990, p. 498.

SECTION 2.6 NONCLINICAL SUMMARY

Section 2.6.7 — Toxicology Tabulated Summary

EMTRICITABINE/RILPIVIRINE/ TENOFOVIR DISOPROXIL FUMARATE

FIXED-DOSE COMBINATION

Gilead Sciences International Limited

17 August 2010

CONFIDENTIAL AND PROPRIETARY INFORMATION

Emtricitabine/Rilpivirine/Tenofovir Disoproxil Fumarate Section 2.6.7 Toxicology Tabulated Summary Final

CONFIDENTIAL Page 2 17AUG2010

TABLE OF CONTENTS

SECTION 2.6 NONCLINICAL SUMMARY ......................................................................................................1 TABLE OF CONTENTS .......................................................................................................................................2 2.6. NONCLINICAL SUMMARY.................................................................................................................7

2.6.7. TOXICOLOGY TABULATED SUMMARY...........................................................................7 2.6.7.1. Toxicology: Overview ............................................................................................8 2.6.7.2. Toxicokinetics: Overview of Toxicokinetic Studies.............................................32

2.6.7.2.A. Emtricitabine ...................................................................................32 2.6.7.2.B. Rilpivirine........................................................................................33 2.6.7.2.C. Tenofovir disoproxil fumarate .........................................................36 2.6.7.2.D. Emtricitabine/Tenofovir DF ............................................................37 2.6.7.2.E. Emtricitabine/Rilpivirine/Tenofovir DF..........................................38

2.6.7.3. Toxicokinetics: Overview of Toxicokinetics Data ..............................................39 2.6.7.3.A. Emtricitabine ...................................................................................39 2.6.7.3.B. Rilpivirine........................................................................................40 2.6.7.3.C. Tenofovir disoproxil fumarate .........................................................42

2.6.7.4. Toxicology: Drug Substance ...............................................................................43 2.6.7.4.A. Emtricitabine ...................................................................................43 2.6.7.4.B. Rilpivirine........................................................................................54 2.6.7.4.C. Tenofovir disoproxil fumarate .........................................................58 2.6.7.4.D. Tenofovir disoproxil fumarate (HPLC Impurity

Profile).............................................................................................62 2.6.7.4.E. Ground TDF/FTC Tablets ...............................................................66 2.6.7.4.F. Ground FTC/RPV/TDF Tablets ......................................................67

2.6.7.5. Single-Dose Toxicity ............................................................................................68 2.6.7.5.A. Emtricitabine ...................................................................................68 2.6.7.5.B. Rilpivirine........................................................................................69 2.6.7.5.C. Tenofovir disoproxil fumarate .........................................................70

2.6.7.6. Repeat-Dose Toxicity: Nonpivotal Studies..........................................................71 2.6.7.6.A. Rilpivirine........................................................................................71 2.6.7.6.B. Tenofovir disoproxil fumarate .........................................................81

2.6.7.7. Repeat-Dose Toxicity: Pivotal Studies ................................................................82 2.6.7.7.A. IUW00701: 14 Day Oral (Gavage) Toxicity Study

in Mice Given FTC..........................................................................82 2.6.7.7.B. TOX599: A Thirty-Day Oral Toxicity Study in

Mice Given 524W91 .......................................................................84 2.6.7.7.C. TOX022: Six Month Oral (Gavage) Toxicity

Study in Mice Given FTC with a 3-Month Interim Kill...................................................................................................86

2.6.7.7.D. TOX628: A Six Month Oral Toxicity Study (With a Three Month Interim Sacrifice) in Mice Given 524W91 ...........................................................................................88

2.6.7.7.E. TMC278-NC119: Three-Month Repeated Dose Oral Toxicity Study in the Mouse ...................................................90

2.6.7.7.F. M990203: A 13-Week Oral Gavage Toxicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Mouse...............................................97

2.6.7.7.G. TOX097: A 3-Month Oral Gavage Study for Bioassay Dose Selection in CD Rats (Emtricitabine).................................................................................99



2.6.7.7.H. TMC278-Exp5692: One-Month Repeated Dose Oral Toxicity Study in the Rat.......................................................101

2.6.7.7.I. TMC278-NC101: Six-Month Repeated Dose Oral Toxicity Study with 1-Month Recovery in the Rat........................103

2.6.7.7.J. 98-TOX-4331-004: A 14-Day Oral Gavage Toxicity Study of bis-POC PMPA Fumarate (GS-4331-05, PMPA Prodrug) in the Albino Rat .................................109

2.6.7.7.K. 96-TOX-4331-003: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 in the Albino Rat ..........................112

2.6.7.7.L. 97-TOX-4331-002: A 13- and 42-Week Oral Gavage Toxicity Study (with a 13-Week Recovery Period) of Bis-POC PMPA (GS-4331-05) in the Albino Rat .....................................................................................114

2.6.7.7.M. TX-164-2001: A 14-Day Oral Gavage Toxicity Study Comparing Non-Degraded and Degraded TDF/FTC in Sprague-Dawley Rats ...............................................118

2.6.7.7.N. TMC278-Exp5650: One-Month Repeated Dose Oral Toxicity Study in the Dog with 1-Month Recovery........................................................................................120

2.6.7.7.O. TMC278-NC115: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog ................................................................................................123

2.6.7.7.P. TMC278-NC107: Twelve-Month Repeated Dose Oral Toxicity Study in the Dog .....................................................131

2.6.7.7.Q. 96-TOX-4331-004: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 in the Beagle Dog.........................135

2.6.7.7.R. 98-TOX-4331-003: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 (bis-POC PMPA; PMPA Prodrug) in the Beagle Dog ...............................................137

2.6.7.7.S. 97-TOX-4331-001: A 13- and 42-Week Oral Gavage Toxicity Study (with 13-Week Recovery Period) of Bis-POC PMPA (GS-4331-05) in the Beagle Dog ....................................................................................139

2.6.7.7.T. TX-164-2004: 4-Week Oral Gavage Toxicity and Toxicokinetic Study with Emtricitabine/Tenofovir Disoporxil Fumarate (FTC/TDF) in Male Dogs with a 4-Week Recovery Period ....................................................143

2.6.7.7.U. TOX600: Repeat-Dose Toxicity: A 30-Day Oral Toxicity Study in Cynomolgus Monkeys Given 524W91 .........................................................................................145

2.6.7.7.V. TOX627: Repeat-Dose Toxicity: A 3-Month Oral Toxicity Study in Cynomolgus Monkeys Given 524W91 .........................................................................................147

2.6.7.7.W. TOX032: Repeat-Dose Toxicity: 52-Week Oral Toxicity Study of TP-0006 (Emtricitabine) in Cynomolgus Monkeys with a 4-Week Recovery Period.............................................................................................149

2.6.7.7.X. TMC278-NC248: Endocrinological Oral (Gavage) 8-week Study in the Female Sexually Immature Cynomolgus Monkey ....................................................................151

2.6.7.8. Genotoxicity: In Vitro.........................................................................................154 2.6.7.8.A. 18637-0-409R: Mutagenicity Test with FTC in the

Salmonella – Escherichia Coli/Mammalian-



Microsome Reverse Mutation Assay with a Confirmatory Assay.......................................................................154

2.6.7.8.B. MUT203: Salmonella/Mammalian-Microsome Assays with 524W91 (Ames Test) ................................................161

2.6.7.8.C. K01-3154: Mutagenicity Test of FTC Using Microorganisms .............................................................................165

2.6.7.8.D. TOX012: In Vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay) ....................................168

2.6.7.8.E. TMC278-Exp5540: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium ................................171

2.6.7.8.F. TMC278-Exp5693: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium ................................174

2.6.7.8.G. TMC278-NC335: In Vitro Bacterial Reverse Mutation Test with R314585 (TMC278.HCl) in Salmonella typhimurium ................................................................177

2.6.7.8.H. TMC278-NC279: In Vitro Bacterial Reverse Mutation Test of TMC278 in Salmonella typhimurium with human liver S9-mix..........................................181

2.6.7.8.I. TMC278-Exp5539: In Vitro Mammalian Forward Mutation Test with L5178Y Mouse Lymphoma Cells (TK-locus) using the Microtiter® Fluctuation Technique ...................................................................187

2.6.7.8.J. TMC278-NC336: Mutation at the Thymidine Kinase (tk) Locus of Mouse Lymphoma L5178Y Cells (MLA) using the Microtitre® Fluctuation Technique ......................................................................................191

2.6.7.8.K. 96-TOX-4331-005: Mutagenicity Test with GS-4331-05 in the Salmonella-Escherichia Coli Mammalian Microsome Reverse Mutation Assay.........................194

2.6.7.8.L. 97-TOX-1278-003: Mutagenicity Test with GS-1278 (PMPA) Lot No. , GS-1278 (PMPA) Lot No. , GS-4331-05 (PMPA prodrug, bis-POC PMPA) Lot No. ( ) in the Salmonella/Mammalian-Microsome Reverse Mutation Assay.............................................197

2.6.7.8.M. K01-3037: Mutagenicity Test of Tenofovir DF Using Microorganisms ..................................................................200

2.6.7.8.N. 97-TOX-4331-007: Mutagenicity Test on GS-4331-05 in the L5178Y TK “+/-” Mouse Lymphoma Forward Mutation Assay ............................................203

2.6.7.8.O. TX-164-2002: Bacterial Reverse Mutation Assay with Emtricitabine/Tenofovir Disproxil Fumarate ........................207

2.6.7.8.P. TX-164-2003: In Vitro Mammalian Cell Gene Mutation Test (L5178Y/TK+/- Mouse Lymphoma Assay) with Emtricitabine/Tenofovir Disoproxil Fumarate ........................................................................................210

2.6.7.9. Genotoxicity (In Vivo)........................................................................................213 2.6.7.9.A. TOX011: In Vivo Mammalian Erythrocyte

Micronucleus Test .........................................................................213 2.6.7.9.B. TMC278-Exp5538: In Vivo Micronucleus Test on

Bone Marrow Cells of Mice ..........................................................215 2.6.7.9.C. 97-TOX-4331-008: Mutagenicity Test on GS-

4331-05 in the In Vivo Mouse Micronucleus Assay .............................................................................................217



2.6.7.9.D. 23291-0-494-OECD: In Vivo/In Vitro Unscheduled DNA Synthesis in Rat Primary Hepatocyte Cultures at 2 Timepoints.............................................218

2.6.7.10. Carcinogenicity...................................................................................................220 2.6.7.10.A. TOX109: TP-0006: Two-Year Oral Oncogenicity

Study in CD-1 Mice.......................................................................220 2.6.7.10.B. TOX108: TP-0006: Two-Year Oral Oncogenicity

Study in CD Rats ...........................................................................225 2.6.7.10.C. TMC278-NC120: Carcinogenicity Study by Oral

Gavage Administration to CD-1 Mice for 104 Weeks ............................................................................................233

2.6.7.10.D. TMC278-NC123: Carcinogenicity Study by Oral Gavage Administration to CD Rats for 104 Weeks.......................236

2.6.7.10.E. M990205: Carcinogenicity: An Oral Carcinogenicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Mouse.............................242

2.6.7.10.F. R990204: An Oral Carcinogenicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Rat................................................................................251

2.6.7.11. Reproductive and Developmental Toxicity: Nonpivotal Studies.......................258 2.6.7.11.A. Emtricitabine .................................................................................258 2.6.7.11.B. Rilpivirine......................................................................................259 2.6.7.11.C. Tenofovir disoproxil fumarate .......................................................260

2.6.7.12. Reproductive & Developmental Toxicity: Fertility & Early Embroyonic Development to Implantation:........................................................261 2.6.7.12.A. TOX036: Study of Fertility and Early Embryonic

Development of TP-0006 Administered by Gavage to CD-1 ® Mice (Segment 1) ...........................................261

2.6.7.12.B. TTEP/95/0028: A Fertility Study in Male Rats Given 524W91 by Gavage.............................................................265

2.6.7.12.C. TMC278-NC124: Oral (Gavage) Male Fertility Study in the Rat .............................................................................269

2.6.7.12.D. TMC278-NC125: Oral (Gavage) Female Fertility Study in the Rat .............................................................................270

2.6.7.12.E. 98-TOX-4331-006: Oral (Gavage) Fertility and General Reproduction Toxicity Study of GS-4331-05 (bis-POC PMPA) in Sprague-Dawley Rats ..............................271

2.6.7.13. Reproductive & Developmental Toxicity: Effects on Embryo-fetal Development.......................................................................................................275 2.6.7.13.A. TOX037: A Study of the Effects of TP-0006

(FTC) on Embryo/Fetal Development in Mice..............................275 2.6.7.13.B. TOX038: A Study of the Effects of TP-0006 on

Embryo/Fetal Development in Rabbits..........................................277 2.6.7.13.C. TMC278-NC105: Oral Developmental Toxicity

Study in the Rats............................................................................279 2.6.7.13.D. TMC278-NC130: Oral Developmental Toxicity

Study in the Rabbit ........................................................................281 2.6.7.13.E. 97-TOX-4331-004: Oral (Gavage) Developmental

Toxicity Study of GS-4331-005 in Rats ........................................283 2.6.7.13.F. 98-TOX-4331-005: Oral (Stomach Tube)

Developmental Toxicity Study of GS-4331-05 in Rabbits...........................................................................................285

2.6.7.14. Reproductive & Developmental Toxicity: Effects on Pre- & Post-Natal Development, Including Maternal Function..............................................287



2.6.7.14.A. TOX039: Pre- and Postnatal Development Study in CD-1® Mice Given TP-0006 by Gavage (Segment III) .................................................................................287

2.6.7.14.B. TMC278-NC168: Oral (Gavage) Pre- and Post-natal Developmental Toxicity and Juvenile Toxicity Dose Range Finding Study in the Rat .............................292

2.6.7.14.C. TMC278-NC131: Oral (Gavage) Pre- and Post-natal Developmental Toxicity Study in the Rat .............................294

2.6.7.14.D. R990202: Oral (Gavage) Developmental and Perinatal/Postnatal Reproduction Toxicity Study of GS-4331-05 (bis-POC PMPA) in Rats Including a Postnatal Behavioral/Functional Evaluation......................................................................................296

2.6.7.15. Studies on Juvenile Animals...............................................................................300 2.6.7.16. Local Tolerance ..................................................................................................301

2.6.7.16.A. Rilpivirine......................................................................................301 2.6.7.16.B. Tenofovir DF .................................................................................302

2.6.7.17. Other Toxicity Studies ........................................................................................303 2.6.7.17.A. Emtricitabine .................................................................................303 2.6.7.17.B. Rilpivirine......................................................................................306 2.6.7.17.C. Tenofovir DF .................................................................................312

2.6.7.18. References ..........................................................................................................321



2.6. NONCLINICAL SUMMARY

2.6.7. TOXICOLOGY TABULATED SUMMARY

2.6.7.1 Toxicology Overview Test Article: Emtricitabine, Rilpivirine, or Tenofovir disoproxil fumarate

Type of Study

Species and Strain

Method of Administration

Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number

Footnotes: GD = gestation day, LD = lactation day, CA = citric acid, HPMC = hydroxylpropyl methylcellulose, PEG400 = polyethylene glycol 400; GLP = Good Laboratory Practice Method of administration: D = divided doses spaced by approximately six hours; SC = subcutaneous - = pre coitum, + = post coitum, F = Female, M = Male a Unless otherwise stated. For Repeat Dose Toxicity, the highest No Observed Adverse Effect Level (NOAEL) is underlined. b Treatment of males 28 days before mating, treatment of females 15 days before mating to day 7 of presumed gestation. CONFIDENTIAL Page 8 17AUG2010

2.6.7.1. Toxicology: Overview Single Dose Toxicity of FTC Mice, Charles

River CD-1 Oral/Gavage Single 4000 Yes Burroughs

Wellcome, USA TTEP/93/0020

Mice, Charles River CD-1

IV Single 200 Yes Burroughs Wellcome, USA

TTEP/93/0023

Rat, Charles River CD

Oral/Gavage Single 4000 Yes Burroughs Wellcome, USA

TTEP/93/0021


IV Single 200 Yes Burroughs Wellcome, USA

TTEP/93/0024

Single Dose Toxicity of RPV Rat, Sprague

Dawley Oral/Gavage Single dose Base: 800

(PEG400) No J&J PRD TMC278-

EXP5559 Dog, Beagle Oral/Gavage Single dose Base: 40, 80

(PEG400), 80 (PEG400 + CA)

No J&J PRD TMC278-EXP5461

Single Dose Toxicity of TDF Rat, Sprague

Dawley Oral/Gavage Single 0, 160, 500, 1500 Yes

R990200

Dog, Beagle Oral/Gavage Single 0, 30, 90, 270 Yes

D990201


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Repeat Dose Toxicity of FTC

Mouse, Charles River CD-1

Oral/Gavage

(D)

2 weeks 0, 120, 600, 3000 Yes , UK

IUW00701


Oral/Gavage 1 month 0, 120, 600, 3000 Yes Burroughs Wellcome, USA

TOX599


Oral/Gavage 6 months 0, 167, 500, 1500 Yes , UK

TOX022


Oral/Gavage 6 months 0, 120, 600, 3000 Yes Burroughs Wellcome, USA

TOX628

Rat, CD Oral/Gavage 3 months 0, 120, 600, 3000 Yes , USA

TOX097

Monkey, Cynomolgus

Oral/Gavage

(D)

1 month 0, 80, 400, 2000 Yes ,

USA

TOX600

Monkey, Cynomolgus

Oral/Gavage

(D)

3 months 0, 40, 200, 1000 Yes ,

USA

TOX627

Monkey, Cynomolgus

Oral/Gavage

(D)

1 year 0, 50, 200, 500

Yes , USA

TOX032


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Repeat Dose Toxicity of RPV

Mouse, Albino Swiss

Oral/Gavage or Diet

2 weeks Hydrochloride salt: 0 (control diet), 40 (diet), 400 (diet), 5000 (diet), 400 (gavage), 2000 (gavage)

No J&J PRD TMC278-NC118

Mouse, CB6F1-nonTgrasH2

Oral/Gavage 1 month Hydrochloride salt: 0 (vehicle), 20, 80, 320 (0.5% HPMC)

Yes TMC278-NC121

Mouse, Albino Swiss

Oral/Gavage 3 months Hydrochloride salt: 0 (vehicle), 20, 80, 320 (0.5% HPMC)

Yes J&J PRD TMC278-NC119

Rat, Sprague Dawley

Oral/Gavage 5 days Base: 0 (water), 0 (vehicle), 40, 400 (PEG400)

No J&J PRD TMC278-TOX5463

Rat, Sprague Dawley

Oral/Gavage 2 weeks Base: 0 (water), 0 (vehicle), 40, 120, 400 (PEG400 + CA)

Yes J&J PRD TMC278-TOX5535

Rat, Sprague Dawley

Oral/Gavage 2 weeks Hydrochloride salt: 0 (vehicle), 400, 1500, 2000 (0.5% HPMC)



Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Rat, Sprague Dawley

Oral/Gavage or Diet

2 weeks Hydrochloride salt: 0 (control diet), 400 (diet), 1200 (diet), 400 (gavage) Base: 400 (diet)


Rat, Sprague Dawley

Oral/Gavage 1 month Base: 0 (vehicle), 10, 400 (PEG400 + CA) Hydrochloride salt: 0 (vehicle), 10, 400 (0.5% HPMC)


Rat, Sprague Dawley

Oral/Gavage 1 month Base: 0 (water), 0 (vehicle), 10, 40, 160 (PEG400 + CA)

Yes

TMC278-EXP5692

Rat, Sprague Dawley

Oral/Gavage 6 months Base: 0 (vehicle), 40, 120, 400 (PEG400 + CA)


Rabbit, New Zealand White

Oral/Gavage 5 days Base: 0 (vehicle), 100, 300, 1000 (0.5% HPMC)


Dog, Beagle Oral/Gavage 7 days Base: 0 (water), 0 (vehicle), 20, 40, 80 (PEG400 + CA)

Yes J&J PRD TMC278-EXP5534


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Dog, Beagle Oral/Gavage 1 month Base: 0 (water), 0 (vehicle), 5, 10, 40 (PEG400 + CA)

Yes J&J PRD TMC278-EXP5650

Dog, Beagle Oral/Gavage 1 month Base: 0 (vehicle), 5, 40 (PEG400 + CA) Hydrochloride salt: 0 (vehicle), 5, 40 (0.5% HPMC)


Dog, Beagle Oral/Gavage 6 months Base: 0 (vehicle), 5, 10, 40 (PEG400 + CA)


Dog, Beagle Oral/Gavage 12 months Base: 0 (vehicle), 5, 10, 40 (PEG400 + CA)

Yes

TMC278-NC107

Monkey, Cynomolgus

Oral/Gavage 8 weeks

Hydrochloride salt: 0 (vehicle), 200, 500 bid (1% HPMC + 0.5% polysorbate 20)

Yes TMC278-NC248

Repeat Dose Toxicity of TDF Mouse, ICR CD-1 Oral/Gavage 13 days 0, 100, 300, 1000 No Gilead Sciences M990191 Mouse, Swiss Crl:

CD-1 (IBR) BR Oral/Gavage 13 weeks 0, 100, 300, 600,

1000 Yes

M990203

Rat, Sprague Dawley

Oral/Gavage 5 days 0, 25, 100, 400 No Gilead Sciences 96-TOX-4331-002


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Rat, Sprague Dawley

Oral/Gavage 14 days 0, 500, 750, 1000, 1250

Yes

98-TOX-4331-004

Rat, Sprague Dawley

Oral/Gavage 28 days 0, 20, 100, 500 Yes

96-TOX-4331-003

Rat, Sprague Dawley

Oral/Gavage 13 or 42 weeks

0, 30, 100, 300, 1000

Yes

97-TOX-4331-002

Dog, Beagle Oral/Gavage 5 days 0, 9, 45, 180 No

96-TOX-4331-001

Dog, Beagle Oral/Gavage 28 days 0, 3, 10, 30 Yes

96-TOX-4331-004

Dog, Beagle Oral/Gavage 28 days 0, 15, 30, 60, 210, bid, qd, q2d, q7d

Yes

98-TOX-4331-003

Dog, Beagle Oral/Gavage 13 or 42 weeks

0, 3, 10, 30 Yes

97-TOX-4331-001

Repeat Dose Toxicity of FTC/TDF Rat, SD Oral/Gavage 14 days 30/20, 100/67,

300/200 Yes

TX-164-2001

Dog, Beagle Oral/Gavage 28 days 2/3, 20/30 Yes , USA

TX-164-2004

Genotoxicity of FTC In vitro non-mammalian cell system

Salmonella typhi. (TA98, TA100, TA1535, TA1537) E. coli (WP2uvrA), (±S9)

In vitro 100 to 5000 g/plate

Yes , USA

18637-0-409R


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Salmonella typhi. (TA98, TA100, TA1535, TA1537, TA1538), (±S9)

In vitro 6.67 to 5000 g/plate

Yes ,

USA

MUT203

Salmonella typhi. (TA98, TA100, TA1535, TA1537) E. coli (WP2uvrA), (±S9)

In vitro up to 5000 g/plate

Yes , Japan

K01-3154

In vitro mammalian cell system

L5178Y mouse lymphoma cells TK+/-, (±S9)

In vitro 1000 to 5000 g/mL

Yes ,

USA

TOX012

In vivo micronucleus Mouse, ICR Oral/Gavage Single 0, 500, 1000, 2000 Yes ,

USA

TOX011

Genotoxicity of RPV

In vitro nonmammalian cell system

Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA102

In vitro Base: 0, 7.81, 15.63, 31.25, 62.50, 125, 250 and 500 μg/plate

Yes J&J PRD TMC278-Exp5540


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number



In vitro Base: 0, 7.81, 15.63, 31.25, 62.50, 125, 250 and 500 μg/plate



In vitro Base: 0, 3, 7.81, 10, 15.63, 25, 31.25, 50, 62.5, 100, 125, 200, 250, 400 and 500 μg/plate



In vitro Hydrochloride salt: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 μg/plate



Mouse Lymphoma cells L5178Y

In vitro Base: 0, 0.5, 1, 2, 2.5, 4, 5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 250, 500 μg/mL



In vitro Hydrochloride salt: 0, 2.5, 5, 10, 12.5, 15, 20, 25, 30, 35, 40 and 50 g/mL

Yes TMC278-NC336


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


In Vivo micronucleus Mouse, Albino Swiss

Oral/Gavage Single dose Base: 0 (vehicle), 100, 400, 1600 (PEG400 + CA)


Genotoxicity of TDF

In vitro non-mammalian cell system

Salmonella typhi. (TA98, TA100, TA1535,) E. coli (WP2uvrA), (±S9)


Yes

96-TOX-4331-005

Salmonella typhi. (TA1535), (±S9)


Yes

97-TOX-1278-003


In vitro Up to 5000 g/plate

Yes , Japan

K01-3037


L5178Y TK +/- Mouse lymphoma Forward Mutation Assay

In vitro 12.5 to 150 g/mL Yes

97-TOX-4331-007

In Vivo Micronucleus Assay

Mouse Crl, Oral gavage Single 0, 500, 1000, 2000 Yes

97-TOX-4331-008

In Vivo Examination of Hepatocytes Unscheduled DNA Synthesis (UDS)

Rat, Fischer 344 Oral gavage Single 0, 500, 1000, 2000 Yes

23291-0-494OECD


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Genotoxicity of FTC/TDF

In vitro non-mammalian cell system


In vitro Fixed ratio of 2 parts FTC to 3 parts TDF up to 5000 g/plate

Yes , USA

TX-164-2002


L5178Y mouse lymphoma cells TK+/-, (±S9)

In vitro Fixed ratio of 2 parts FTC to 3 parts TDF, 15 to 500 g/mL

Yes , USA

TX-164-2003

Carcinogenicity of FTC

Mouse, CD-1 (ICR) Oral/Gavage 2 years 0, 80, 250, 750 Yes , USA

TOX109

Rat, CD (Sprague Dawley)

Oral/Gavage 2 years 0, 60, 200, 600 Yes , USA

TOX108

Carcinogenicity of RPV Mouse, CD-1 Oral/Gavage 2 years Hydrochloride

salt: 0 (vehicle), 20, 60, 160 (0.5% HPMC)

Yes TMC278-NC120

Rat, Sprague Dawley

Oral/Gavage 2 years Hydrochloride salt: 0 (vehicle), 40, 200, 500, 1500 (0.5% HPMC)

Yes TMC278-NC123


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Carcinogenicity of TDF

Mouse, CD-1 (ICR) Oral/Gavage 2 years 0, 100, 300 600 Yes

M990205

Rat, CD (Sprague Dawley)

Oral/Gavage 2 years 0, 30, 100, 300 Yes

R990204

Reproductive and Developmental Toxicity of FTC Fertility and Early Embryonic Development

Mouse, CD-1 Oral/Gavage (D)

F: 2 wks to +6 days M: 4 wks to +6 wks

0, 250, 500, 1000

Yes

, USA

TOX036

Rat, CD Sprague-Dawley (males only)

Oral/Gavage M: 73 days + throughout mating

0, 150, 750, 3000 Yes Burroughs Wellcome, USA

TTEP/95/0028

Embryo-fetal Development


GD6 15 0, 250, 500, 1000 Yes , USA

TOX033


GD6 15 0, 250, 500, 1000 Yes , USA

TOX037


Oral/Gavage (D)

GD7 19 0, 250, 500, 1000 Yes , USA

TOX034


Oral/Gavage (D)

F: GD7 19 0, 100, 300, 1000 Yes , USA

TOX038


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Pre-and Postnatal Development Including Maternal Function


F0 F dosed only: from GD6 to LD 20

0, 250, 500, 1000 Yes

, USA

TOX039

Reproductive and Developmental Toxicity of RPV Fertility and Early Embryonic Development

Rat, Sprague Dawley

Oral/Gavage 10 weeks prior to mating and 4 weeks after the mating period

Hydrochloride salt: 0 (vehicle), 100, 400, 1600 (0.5% HPMC)

Yes TMC278-NC124

Fertility and Early Embryonic Development

Rat, Sprague Dawley

Oral/Gavage 2 weeks prior to pairing and up to day 7 of pregnancy




Rat, Sprague Dawley

Oral/Gavage GD6 17 Base: 0 (vehicle), 40, 120, 400 (PEG400 + CA)


Rat, Sprague Dawley

Oral/Gavage GD6 17 Base: 0 (vehicle), 40, 120, 400 (PEG400 + CA)



Oral/Gavage GD6 19 Base: 0 (vehicle), 25, 75, 150 (0.5% HPMC)



Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number



Rabbit, New Zealand white



Rabbit, New Zealand white



Prenatal & Postnatal Development and Juvenile Toxicity

Rat, Sprague Dawley

Oral/Gavage GD6 LD7 (dams), Day 12 to Day 25 of age (pups)


No TMC278-NC168

Prenatal & Postnatal Development

Rat, Sprague Dawley

Oral/Gavage GD6 LD20 Hydrochloride salt: 0 (vehicle), 40, 120, 400 (0.5% HPMC)

Yes TMC278-NC131

Reproductive and Developmental Toxicity of TDF Fertility & Early Embryonic Development

Rat, Sprague Dawley

Oral gavage b 0, 100, 300, 600 Yes

98-TOX-4331-006


Rat, Sprague Dawley

Oral gavage F: GD7 17 0, 50, 150, 450

Yes

97-TOX-4331-004


Oral gavage F: GD6 18 0, 30, 100, 300

Yes

98-TOX-4331-005

Prenatal & Postnatal Development

Rat, Crl: CD (SD) IGS BR VAF/Plus

Oral gavage F: GD7 LD20or F: GD7 24

0, 50, 150, 450, 600

Yes

R990202


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Local Tolerance of RPV Bovine corneal opacity - permeability eye irritation test

Bovine corneas In Vitro Hydrochloride salt: 20% w/w in 0.9% NaCl (vehicle)


Phototoxicity Balb/c mouse fibroblast

In vitro Hydrochloride salt: 0.001 to 2.16 mg/L


Primary skin irritation Rabbit, New Zealand White

Dermal application

4 hours Hydrochloride salt: 0.5 g

No TMC278-NC159

Local lymph node assay

Mouse, CBA/Ca/Ola/Hsd

Dermal application

3 days Hydrochloride salt: 5, 10 and 35% in Acetone/Olive Oil (4:1)

No TMC278-NC199

Local Tolerance of TDF Primary eye irritation study


Topical Single dose 0.1 mL (0.0760g) Yes

B990165

Primary skin irritation study


Topical Single dose 0.5 g Yes

B990166


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Other Studies with FTC

Immunotoxicity FTC - Immunotoxicity in rats

Rat, Sprague Dawley

Oral gavage 28 days 0, 60, 240, 1000 Yes ,

Scotland

TOX146

Impurities Mouse Oral Qualification Study

Mouse, CD-1

Oral gavage 1 month 50/1, 150/3 and 450/9

Yes , USA

TOX153

Oral Qualification Study of TP-0006 Produced by the

Process

Mouse, CD-1

Oral gavage 1 month 0, 50, 150, 450 Yes , USA

TX-162-2001

FTC CHO ( S9) In vitro 313 to 5000 g/mL, 1% TP-

0296

Yes , USA

TOX152

FTC Salmonella typhi. (TA98, TA100, TA1535, TA1537) E. coli (WP2uvrA), ( S9)

In vitro 1.5 to 5000 g/plate and 1%

TP-0296, up to 50 g/plate

Yes , USA

TOX151


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Other Studies with RPV Endocrinology In Vitro Guinea Pig Adrenal Cell Test for the evaluation of the cortisol biosynthesis

Guinea pig adrenal cells

In vitro Base: 10-9 to 10- 5M (0.00037 to 3.7

g/mL)


A study on the effect of R278474 on the biosynthesis of cortisol in crude subcellular fractions of dog adrenal cortex

Dog adrenal cortex In vitro Base: 0.03 to 75 M (0.011 to

27.75 g/mL)

No J&J PRD TMC278-FK4790

Single Dose Oral Endocrinology Study in the Beagle Dog

Dog, Beagle Oral/Gavage Single dose Base: 0 (vehicle), 20, 80 (PEG400 + CA)


Impurities In vitro bacterial reverse mutation test with R314585 (TMC278.HCl) spiked with 4% ( .HCl) in Salmonella typhimurium


In vitro Hydrochloride salt: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 g/plate spiked with 4%


不純物B*

不純物B'*

不純物B*



Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


In vitro bacterial reverse mutation test with R314585 (TMC278.HCl) spiked with 4% ( .HCl) in Salmonella typhimurium


In vitro Hydrochloride salt: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 g/plate spiked with 4%


Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y (MLA) using the Microtitre fluctuation technique


In vitro Hydrochloride salt: up to 75 g/mL spiked with 4%

Yes TMC278-NC311

Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) using the Microtitre fluctuation technique


In vitro Hydrochloride salt: up to 75 g/mL spiked with 4%

Yes TMC278-NC312

不純物C*

不純物C'*

不純物B*

不純物C*

不純物C*



Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


1-Month Repeated Dose Oral Impurity Qualification Study for TMC278.HCL in the Rat

Rat, Sprague Dawley

Oral/Gavage 1-month Hydrochloride salt: 0 (vehicle), 10, 10 spiked with 4%

, 10 spiked with 4% (0.5% HPMC)


In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium


In vitro : 0 (vehicle), 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000

g/plate




In vitro : 0 (vehicle), 78.13, 156.25, 312.5, 625, 1250, 2500 and 500 g/ plate


不純物B*

不純物C*

不純物D'*

不純物D'*



Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number




In vitro : Exp 1 (2): 0 (vehicle), 78.13 (93.75), 156.25 (187.5), 312.5 (375), 625 (750), 1250 (1500), 2500 (3000) and 5000 g/ plate

Yes J&J PRD TMC278-1646_0016315

Screening Chromosome Aberration Test in Human Lymphocyes In Vitro

Human lymphocytes

In vitro 4 or 24 hours : 0, 65.22, 130.44, 260.88 and 521.75 g/mL

No

TMC278-NC184



In vitro : Experiment 1 (2): 0 (vehicle), 78.13 (93.75), 156.25 (187.5), 312.5 (375), 625 (750), 1250 (1500), 2500 (3000) and 5000 g/plate

Yes J&J PRD TMC278-1646_0015298

不純物D'*

不純物D'*

不純物E*



Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number




In vitro : Experiment 1 (2): 0 (vehicle), 78.13 (93.75), 156.25 (187.5), 312.5 (375), 625 (750), 1250 (1500), 2500 (3000) and 5000 g/plate

Yes J&J PRD TMC278-1646_0015299

不純物F*



Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Other Toxicity Studies for TDF/Tenofovir

Antigenicity TDF: Dermal sensitisation study

Guinea pig, Hartley

Topical 5 weeks: 3 wks induction, 1 wk challenge & 1 wk re-challenge

75% w/v q7d, for 3 wks, 50% w/v or 100% for challenge & 65%w/v & for re-challenge

Yes

G990167

Mechanistic Studies (TDF/Tenofovir) TDF: Interaction study/ mitochondrial toxicity

Rat, Sprague Dawley

Oral/Gavage 28 days 0, 40 (adefovir dipivoxil), 300 (TDF) & 40/300 adefovir dipivoxil/TDF

No Gilead Sciences R2000096

TDF: Mitochondrial toxicity

Woodchuck (Marmota monax)

Oral/Gavage 90 days 0, 15, 50/TDF No (in life phase)

W2000042

Tenofovir: Toxicity to osteoblasts

Human osteoblasts-like (NHOst) cells

In vitro 3 weeks 10 g/mL No Gilead Sciences V2000122

TDF: Effects on serum/ urine phosphate levels

Rat, Sprague Dawley

Oral/Gavage 3 days 0, 400 No Gilead Sciences R2000095

TDF: Effects on serum/ urine phosphate levels

Rat, Sprague Dawley

Oral/Gavage or IV

3 day Tenofovir: 50 IV, tenofovir 180 PO, TDF: 400 PO

No Gilead Sciences R2000099

TDF: Effects on serum phosphorous & Ca concentrations

Rat, Sprague Dawley

Oral/Gavage 6 days 0, 400 No Gilead Sciences R2000043


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


TDF: Effects on bone mass

Rat, Sprague Dawley

Oral.Gavage 28 days 0, 40, 400 Yes

R2000036

TDF: Effects on serum phosphorous & Ca concentrations

Monkey, Rhesus Oral/Gavage & SC

56 days TDF 0, 30, 250, 600, PO Tenofovir 30 SC.

Yes (in-

life phase)

P2000078

Impurities TDF: Metabolites & Impurities

Rat, Sprague Dawley

Oral/Gavage 14 days 0, 30, 100, 300 Yes

R2000081


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Genotoxicity In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium:

S typhimurium & EColi Mammalian Microsome Reverse Mutation Assay

In vitro - 92.2 to 4610 μg/plate +/- metabolic activation & 100 to 5000 μg/plate ± metabolic activation

Yes

95-TOX-1278-006

Tenofovir: L5178Y TK +/- Mouse lymphoma Forward Mutation Assay Tenofovir


In vitro - 78.1 to 5000 μg/mL

Yes

95-TOX-1278-007

Safety Studies in Efficacy Models

Tenofovir: Effects on bone

Monkey, Rhesus SC Gestational Day 80–157; Infants continued through 9 months

0, 30 No

J Acquir Immune Defic Syndr Hum Retrovirol 1999 Apr 1;20 (4):323-33. {1787}

Tenofovir: Effects on cortical bone strength

Monkey, Rhesus SC Followed by in vitro analysis of bone specimens from different studies & experimental regimens

Up to 2 years 0, 30, 30 (with dose reduction to 10).

No

T1278-00030


Type of Study

Species and Strain


Duration of Dosing

Doses (mg/kg)a

GLP Compliance

Testing Facility

Study Number


Tenofovir: Effects on bone

Monkey, Rhesus SC 28 days & 434 days

0, 30 No

T1278-00034

Tenofovir: Effects on kidney and bone

Monkey, Rhesus SC Up to 5 years 0, 30, 30 (with dose reduction to 2.5, 5 or 10)

No

{7311}

2.6.7.2.A. Toxicokinetics Overview of Toxicokinetic Studies Test Article: Emtricitabine Type of Study

Test System

Method of Administration Doses (mg/kg)

GLP Compliance

Study Number


2.6.7.2. Toxicokinetics: Overview of Toxicokinetic Studies 2.6.7.2.A. Emtricitabine

Fourteen Day (Gavage) Study Given FTC Mouse, Charles River CD-1

Oral gavage 0, 120, 600, 3000 Yes IUW00701 Appendix 3

Thirty Day Study Given 524W91 Mouse, Charles River CD-1

Oral gavage 0, 120, 600, 3000 Yes TOX599 Appendix 5

Six Month (Gavage) Given FTC With a 3 Month Interim Kill


Oral gavage 0, 167, 500, 1500 Yes TOX022 Addendum

A Six Month Study (with a 3 Month Interim Sacrifice) Given 524W91


Oral gavage 0, 120, 600, 3000 Yes TOX628 Appendix F

TP-0006: Two year Oral Oncogenicity Study in CD-1 Mice


Oral gavage 0, 80, 250, 750 Yes TOX109

A 3 Month Gavage for Bioassay Dose Selection

Rat, CD Oral gavage 0, 120, 600, 3000 Yes TOX097 Appendix N

TP-0006: Two Year Oral Oncogenicity Study in CD Rats

Rat, CD Oral gavage 0, 60, 200, 600 Yes TOX108

A 30 Day Study in Cynomolgus Monkeys Given 524W91

Monkey, Cynomolgus


A Three Month study Given 524W91 Monkey, Cynomolgus


Toxicokinetic Report for a 52 Week Toxicity Study of FTC (Emtricitabine) With a 4 week Recovery Period

Monkey, Cynomolgus


A Study of the Effects of TP-0006 (FTC) on Embryo/Fetal Development in Mice

Mouse, CD-1 Oral gavage 0, 250, 500, 1000 Yes TOX037

A Study of the Effects of TP-0006 on Embryo/Fetal Development in Rabbits (TOX038)

Rabbits, New Zealand White

Oral gavage 0, 100, 300, 1000 Yes TOX038

2.6.7.2.B. Toxicokinetics Overview of Toxicokinetic Studies Test Article: Rilpivirine Type of Study

Test System


GLP Compliance

Study Number


2.6.7.2.B. Rilpivirine

Single Dose Toxicity Rat, Sprague Dawley

Oral/Gavage Base: 800 (PEG400) No TMC278-EXP5559 (FK4278)

Single Dose Toxicity Dog, Beagle Oral/Gavage Base: 40, 80 (PEG400) 80 (PEG400 + CA)

No TMC278-EXP5461 (FK4102)

2-Week Repeat Dose Toxicity Mouse, Albino Swiss

Oral/Gavage or diet

Hydrochloride salt: 0 (control diet), 40 (diet), 400 (diet), 5000 (diet), 400 (gavage), 2000 (gavage)

No TMC278-NC118

1-Month Repeat Dose Toxicity Mouse, CB6F1-nonTgrasH2

Oral/Gavage Hydrochloride salt: 0 (vehicle), 20, 80, 320 (0.5% HPMC)

Yes TMC278-NC121

3-Month Repeat Dose Toxicity Mouse, Albino Swiss


Yes TMC278-NC119

5-Day Repeat Dose Toxicity Rat, Sprague Dawley

Oral/Gavage Base: 0 (vehicle), 40, 400 (PEG400)

No TMC278-EXP5463 (FK4103)

2-Week Repeat Dose Toxicity Rat, Sprague Dawley

Oral/Gavage Base: 0 (water), 0 (vehicle), 40, 120, 400 (PEG400 + CA)

Yes TMC278-EXP5535 (FK4243)



No TMC278-NC177


Oral/Gavage or diet

Hydrochloride salt: 0 (control diet), 400 (diet), 1200 (diet), 400 (gavage) Base: 400 (diet)

No TMC278-NC136


Test System


GLP Compliance

Study Number


1-Month Repeat Dose Toxicity Rat, Sprague Dawley

Oral/Gavage Base: 0 (vehicle),10, 400 (PEG400 + CA) Hydrochloride salt: 0 (vehicle), 10, 400 (0.5% HPMC)

Yes TMC278-NC117

1-Month Repeat Dose Toxicity and Immunotoxicity Evaluation

Rat, Sprague Dawley

Oral/Gavage Base: 0 (water), 0 (vehicle),10, 40, 160 (PEG400 + CA))

Yes TMC278-EXP5692

6-Month Repeat Dose Toxicity Rat, Sprague Dawley

Oral/Gavage Base: 0 (vehicle), 40, 120, 400 (PEG400 + CA)

Yes TMC278-NC101

5-Day Repeat Dose Toxicity Rabbit, New Zealand white

Oral/Gavage Base: 0 (vehicle), 100, 300, 1000 (0.5% HPMC)

No TMC278-NC126

7-Day Repeat Dose Toxicity Dog, Beagle Oral/Gavage Base: 0 (water), 0 (vehicle), 20, 40,80 (PEG400 + CA)

Yes TMC278-EXP5534 (FK4244)

1-Month Repeat Dose Toxicity Dog, Beagle Oral/Gavage Base: 0 (water), 0 (vehicle), 5, 10, 40 (PEG400 + CA)

Yes TMC278-EXP5650

1-Month Repeat Dose Toxicity Dog, Beagle Oral/Gavage Base: 0 (vehicle), 5, 40 (PEG400 + CA) Hydrochloride salt: 0 (vehicle), 5, 40 (0.5% HPMC)

Yes TMC278-NC116

6-Month Repeat Dose Toxicity Dog, Beagle Oral/Gavage Base: 0 (vehicle), 5, 10, 40 (PEG400 + CA)

Yes TMC278-NC115

12-Month Repeat Dose Toxicity Dog, Beagle Oral/Gavage Base: 0 (vehicle), 5, 10, 40 (PEG400 + CA)

Yes TMC278-NC107

8-Week Repeat Dose Toxicity Monkey, Cynomolgus

Oral/Gavage Hydrochloride salt: 0 (vehicle), 200, 500 bid (1% HPMC + 0.5% polysorbate 20)

Yes TMC278-NC248


Test System


GLP Compliance

Study Number


Genotoxicity - In vivo (Micronucleus assay)

Mouse, Albino Swiss


Yes TMC278-Exp5538 (FK4259)

Carcinogenicity Mouse, CD-1 Oral/Gavage Hydrochloride salt: 0 (vehicle), 20, 60, 160 (0.5% HPMC)

Yes TMC278-NC120

Carcinogenicity Rat, Sprague Dawley

Oral/Gavage Hydrochloride salt: 0 (vehicle), 40, 200, 500, 1500 (0.5% HPMC)

Yes TMC278-NC123

Developmental Toxicity Rat, Sprague Dawley


Yes TMC278-NC105

Developmental Toxicity Rabbit, New Zealand White

Oral/Gavage Base: 0 (vehicle), 5, 10, 20 (0.5% HPMC)

Yes TMC278-NC130

Per- and Post-natal Developmental study with Juvenile Exposure

Rat, Sprague Dawley


No TMC278-NC168

CA = citric acid, GLP = Good Laboratory Practice, HPMC = hydroxylpropyl methylcellulose , PEG400 = polyethylene glycol 400 a unless otherwise stated

2.6.7.2.C. Toxicokinetics Overview of Toxicokinetic Studies Test Article: Tenofovir disoproxil fumarate Type of Study

Test System


GLP Compliance

Study Number


2.6.7.2.C. Tenofovir disoproxil fumarate

Thirteen week toxicity study Mouse Swiss Crl: CD-1 (IBR) BR

Oral/Gavage 100, 300, 600, 1000 Yes M990203-PK

Fourteen day toxicity study Rat, Sprague Dawley

Oral/Gavage 500, 750, 1000, 1250 Yes 98-TOX-4331-004-PK

Twenty-eight day toxicity study Rat, Sprague Dawley

Oral/Gavage 20, 100, 500 Yes 96-TOX-4331-003-PK

Thirteen & forty two week toxicity study Rat, Sprague Dawley

Oral/Gavage 30, 100, 300, 1000 Yes 97-TOX-4331-002-PK

Five day toxicity study Dog, Beagle Oral/Gavage 9, 45, 180 No 96-TOX-4331-001-PK Twenty-eight day toxicity study Dog, Beagle Oral/Gavage 15, 30, 60, 210, bid, qd,

q2d, q7d Yes 98-TOX-4331-003-PK

Twenty-eight day toxicity study Dog, Beagle Oral/Gavage 3, 10, 30 Yes 96-TOX-4331-004-PK Thirteen & forty-two week toxicity study Dog, Beagle Oral/Gavage 3, 10, 30 Yes 97-TOX-4331-001-PK Micronucleus assay Mouse, Crl Oral/Gavage 500, 1000, 2000 Yes 97-TOX-4331-008-PK Carcinogenicity study Rat, Sprague

Dawley Oral/Gavage 30, 100, 300 Yes R990204

Carcinogenicity study Mouse, albino Oral/Gavage 100, 300, 600 Yes M990205 Developmental toxicity study Rabbit, New

Zealand White Oral/Stomach tube 30, 100, 300 Yes 98-TOX-4331-005-PK

Prenatal & postnatal developmental study Rat, Crl: CD (SD) IGS BR VAF/Plus

Oral/Gavage 50, 150, 450, 600 Yes R990202-PK

Mechanistic study Rat, Sprague Dawley

Oral/Gavage 40, 400 Yes R2000036-PK

Mechanistic study Monkey, Rhesus Oral, nasogastric Subcutaneous

30, 250, 600 30

Yes P2000078-PK

Metabolites & Impurities Rat, Sprague Dawley

Oral/Gavage 30, 100, 300 Yes R2000081-PK

2.6.7.2.D. Toxicokinetics Overview of Toxicokinetic Studies Test Article: Emtricitabine and tenofovir disoproxil fumarate

Type of Study

Test System


GLP Compliance

Study Number


2.6.7.2.D. Emtricitabine/Tenofovir DF

Twenty-eight Day toxicity study Dog, Beagle Oral/Gavage 2/3; 20/30 Yes TX-164-2004

2.6.7.2.E. Toxicokinetics Overview of Toxicokinetic Studies Test Article: Emtricitabine, Rilpivirine, and tenofovir disoproxil fumarate


2.6.7.2.E. Emtricitabine/Rilpivirine/Tenofovir DF

Not Applicable

2.6.7.3.A. Toxicokinetics Overview of Toxicokinetics Data Test Article: Emtricitabine


2.6.7.3. Toxicokinetics: Overview of Toxicokinetics Data 2.6.7.3.A. Emtricitabine

Steady State AUC (μg·h/mL) Daily Dose Mouse Rat Monkeymg/kg M F M F M F 40 13.1n 12.2o 14.4n 13.4o

50 16.1p

17.0q 18.1r 18.1p

22.9q 33.2r 60 29.9j 42.9k 30.9j 52.5k 80 25.5f 27.5g 27.6f 23.7g 37.1l 31.6m 37.8l 33.6m

120 169.7e 103a

78b 115.4e 74a

83b 61.6h 65.6i 55.6h 69.3i

167 63.8c 82.3d 67.9c 93.1d

200 97.3j 137k 156j 171k 64.8n 71.0o

74.9p

102.9q 97.2r

54.6n 51.9o 99.9p

119.9q 97.8r 250 91.0f 90.9g 65.0f 91.78g 400 170l 196m 170l 164m

500 181.4c 248.4d 209.6c 284.4d 265.5p 341.7q

274 r 195.7p 321.1q

238r

600 585.7e 499a 410b

490.8e 304a 326b

296h 329i

280j 327k 260h 362i

260j 404k

750 235f 287g 209f 323g 1000 403n 331o 274n 233o 1500 513c 732d 569c 899d 2000 631l 730m 765l 732m

3000 2026e 1936a 1800b

1549e 1516a

1711b 1413h 1276i 1357h 1646i

a = TOX599 (day 32), b = TOX599 (day 3), c = TOX022 (week 13), d = TOX022 (week 26), e= TOX628 (week 26), f = TOX109 (week 2), g = TOX109 (week 26), h = TOX097 (day 2), i = TOX097 (day 90), j = TOX108 (week 2), k = TOX108 (week 26), l = TOX600 (day 3), m = TOX600 (day 27), n = TOX627 (day 87), o = TOX627 (day 3), p = TOX032 (week 13), q = TOX032 (week 26), r = TOX032 (week 52). Note: Cmax, T1/2, Clearance when available are given in the individual study Tables, 2.6.7.7. In a combination study (FTC + TDF) in dogs, AUCs for FTC were 25.28 and 32.33 g*h/mL, respectively, for 20 mg/kg/day FTC alone or in combination with TDF (TX-164-2004).

2.6.7.3.B. Toxicokinetics Overview of Toxicokinetics Data Test Article: Rilpivirine



Cmax at steady state ( g/mL) Mouse Rat Rabbit Dog Monkey Daily Dose

(mg/kg/day) TMC278 Formulation

Study Duration

(days) M F M F F M F F 5 base a 363 1.1 1.5

5 (pregnant) base b 14 (GD19) 6.7 10 base a 363 1.3 2.2

10 (pregnant) base b 14 (GD19) 10 20 HCl c Week 28 9.8 9.9

20 (pregnant) base b 14 (GD19) 15 40 HCl d Week 39 0.82 2.1

40 (juvenile) HCl e 14 2.6 5.8 40 base a 363 4.1 5.5

40 (pregnant) base f 11 (GD16) 5.6 40 base g 175 1.7 6.6 60 HCl c Week 28 22 29

120 (juvenile) HCl e 14 3.7 3.6 120 (pregnant) base f 11 (GD16) 7.2

120 base g 175 3 8.8 160 HCl c Week 28 36 58 200 HCl d Week 39 1.3 4.7 200 HCl h 55 0.18

400 (juvenile) HCl e 14 9.1 7.3 400 (juvenile) HCl e 14 6.4 4.9 400 (pregnant) base f 11 (GD16) 13

400 base g 175 6.2 16 500 HCl d Week 39 1.8 8.5 500 HCl h 55 0.31 1500 HCl d Week 39 2.2 9.4

Only the long-term and main studies in mice, rats, rabbits and dogs and cynomolgus monkeys after oral administration of TMC278 are presented in this table. a: NC107; b: NC130; c: NC120; d: NC123; e: NC168; f: NC105; g: NC101 (doses were split and given twice daily with a 1.5-hour interval from Day 84 onwards); h: NC248 (500 mg/kg/day was given as 250 mg/kg/day twice daily with 6-hour interval). GD = Gestation Day

2.6.7.3.B. Toxicokinetics Overview of Toxicokinetics Data Test Article: Rilpivirine


AUC0-24h at steady state ( g.h/ml) Mouse Rat Rabbit Dog Monkey Daily Dose

(mg/kg/day) TMC278 Formulation

Study Duration

(days) M F M F F M F F 5 base a 363 17 19

5 (pregnant) base b 14 (GD19) 105 10 base a 363 24 36

10 (pregnant) base b 14 (GD19) 170 20 HCl c Week 28 76 51

20 (pregnant) base b 14 (GD19) 232 40 HCl d Week 39 6.3 14

40 (juvenile) HCl e 14 12 18 40 base a 363 65 61

40 (pregnant) base f 11 (GD16) 37 40 base g 175 12 50 60 HCl c Week 28 230 278

120 (juvenile) HCl e 14 34 28 120 (pregnant) base f 11 (GD16) 63

120 base g 175 35 116 160 HCl c Week 28 505 766 200 HCl d Week 39 8.2 41 200 HCl h 55 2.7

400 (juvenile) HCl e 14 50 53 400 (juvenile) HCl e 14 43 39 400 (pregnant) base f 11 (GD16) 152

400 base g 175 73 244 500 HCl d Week 39 14 46 500 HCl h 55 4.6

1500 HCl d Week 39 18 84

Only the long-term and main studies in mice, rats, rabbits and dogs and cynomolgus monkeys after oral administration of TMC278 were presented in this table. a: NC107; b: NC130; c: NC120; d: NC123; e: NC168; f: NC105; g: NC101 (doses were split and given twice daily with a 1.5-hour interval from Day 84 onwards); h: NC248

(500 mg/kg/day was given as 250 mg/kg/day twice daily with 6-hour interval). GD = Gestation Day

2.6.7.3.C. Toxicokinetics Overview of Toxicokinetics Data Test Article: Tenofovir disoproxil fumarate

A From a mixed M/F group, B= individual animal Data from studies 1= M990203-PK, 2= 96-TOX-4331-003-PK, 3= 97-TOX-4331-002-PK, 4= 96-TOX-4331-001-PK, 5= 98-TOX-4331-003-PK, NB doses 30mg/kg qd, 60mg/kg q2d, 210 mg/kg q7d, 6= 96-TOX-4331-004-PK, 7=97-TOX-4331-001-PK, 8= 98-TOX-4331-005-PK, 9= R990202-PK, 10= R2000036-PK, 11= P2000078-PK, 12= R2000081-PK, 13=R990204-PK (011295), 14 Median data from Study 901- 300 mg/d. 15= data from M990205. 16= data from TX-164-2004, second value in combination with FTC. Limited data precluded accurate estimation of AUC values from studies 97-TOX-4331-008-PK, 98-TOX-4331-004-PK. CONFIDENTIAL Page 42 17AUG2010


Steady State AUC (μg·h/mL) Daily Dose Mouse Rat Rabbit Dog Monkey Human

mg/kg M F M F M F M F M F M F 3 - - - - - - 2.12 7, A 2.12 7,A - - 3.014 3.014 9 - - - - - - 4.194,B

4.984,B - - -

10 - - - - - - 3.846 6.63 7, A

6.63 7.A - -

30 - - 3.683, , 2.0912

2.7013

3.873, 2.1912

3.0613

- 19.28 14.46, 29.45, 29.07, A

18.4/20.916

24.66, 29.07,A

4.4611 3.6311

40 - - 3.1310 - - - - - - - 45 - - - - - - 30.54,B,

62.94,B - - -

50 - - - 5.889 - - - - - - 60 - - - - - - 67.05 - - -

100 14.61 - 8.433, 4.8212

6.6014

8.253 4.4212

7.4614

- 65.38 - - - -

150 - - - 11.729 - - - - - - 180 - - - - - - 18404,B,

89.14,B - - -

210 - - - - - - 2195 - - - 250 - - - - - - - - 26.911 14.011 300 35.81 - 17.123

10.512

16.6914

18.073, 8.5512

15.7214

- 212.58 - - - -

400 - - 19.610 - - - - - - - 450 - - - 26.769 - - - - - - 500 - - 172 13.72 - - - - - - 600 68.11,

44.215 55.21,

50.915 - 27.299 - - - - 53.311 59.311

750 - - - - - - - - - - 1000 - - 70.23 59.53 - - - - - -

2.6.7.4.A. Toxicology Drug Substance Test Article: Emtricitabine

Note: Footnotes are provided on the last page of the table. CONFIDENTIAL Page 43 17AUG2010

2.6.7.4. Toxicology: Drug Substance 2.6.7.4.A. Emtricitabine

Purity (%) Individual impurities (Specified and Unspecified)

Individual Unspecified Impurities

Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

Dio

xola

ne

(TP-

0297

)

SPECIFICATION: Batch No. 1

.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

e 1.1%a 97.7% AUC

NAb NAb NAb NAb NR NR TOX011 TOX012

f 2.0%a 95.6%g ND 0.33% ND 0.36% 0.08% TP-0270 0.05% TP-0296 0.05% RRT 0.67

0.05% RRT 1.21 0.1% RRT 1.27 0.05% RRT 1.77

0.05% RRT 1.99 0.05% RRT 2.27 0.08% RRT 2.78 0.08% RRT 3.25

1.19% TOX022


不純物FG*

不純物FH*

不純物FI*

不純物FJ*





Total Impurities

(%

) (TP

-027

4)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

e,f 1.0%a 93.0%g ND ND 0.08% 0.26% 0.07% TP-0296 0.09% RRT 0.52 0.12% RRT 0.53 0.10% RRT 0.54 0.06% RRT 0.58

0.05% RRT 0.59 0.05% RRT 0.72 0.10% RRT 0.74 0.16% RRT 0.92

0.05% RRT 0.93 0.83% RRT 1.01 0.13% RRT 1.06 0.06% RRT 1.13 0.09% RRT 1.45 0.10% RRT 1.49 0.05% RRT 1.51 0.24% RRT 1.99

3.04%g TOX022

0.19% RRT 2.27 0.06% RRT 2.29 0.07% RRT 2.39 0.09% RRT 2.42 0.09% RRT 2.47

不純物FG*

不純物FH*

不純物FI*

不純物FJ*






Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

f 1.0%a 96.6% ND ND ND 0.12% 0.05% TP-0270

0.07% TP-0296 0.05% RRT 0.92

0.05% RRT 0.97 0.05% RRT 1.06

0.13% RRT 1.27 0.05% RRT 1.45

0.13% RRT 1.51 0.05% RRT 1.57

0.07% RRT 2.27

0.57% TOX022

不純物FG*

不純物FH*

不純物FI*

不純物FJ*





Individual Unspecified Impurities Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

f 1.0%a 97.3% ND ND ND 0.43% 0.05% TP-0270 0.05% TP-0296 0.06% RRT 0.92

0.05% RRT 0.97 0.05% RRT 1.06 0.05% RRT 1.27 0.05% RRT 1.52

0.06% RRT 2.27

0.60% TOX022

不純物FG*

不純物FH*

不純物FI*

不純物FJ*





Individual Unspecified Impurities Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

e,f 1.4%a 94.3%g ND ND ND 0.24% 0.07% TP-0296 0.05% RRT 0.51

0.10% RRT 0.54 0.05% RRT 0.72

0.06% RRT 0.74 0.16% RRT 0.92

0.05% RRT 0.93 0.96% RRT 1.02 0.11% RRT 1.06

0.05% RRT 1.45 0.05% RRT 1.50

0.05% RRT 1.51 0.29% RRT 1.99 0.14% RRT 2.27 0.06% RRT 2.30 0.09% RRT 2.42

0.05% RRT 2.47

2.34%g TOX022 TOX032

不純物FG*

不純物FH*

不純物FI*

不純物FJ*






Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

e,f 1.7%a 92.9%g ND ND 0.08% 0.26% 0.08% TP-0296 0.09% RRT 0.52 0.12% RRT 0.53 0.10% RRT 0.54 0.06% RRT 0.58 0.05% RRT 0.59 0.05% RRT 0.72 0.11% RRT 0.74 0.17% RRT 0.92

0.05% RRT 0.93 0.83% RRT 1.01 0.14% RRT 1.06 0.10% RRT 1.45 0.10% RRT 1.50 0.05% RRT 1.51 0.25% RRT 1.99 0.19% RRT 2.27 0.06% RRT 2.29 0.07% RRT 2.40 0.10% RRT 2.42 0.09% RRT 2.46

3.13%g TOX036 TOX037 TOX033 TOX034 TOX038 TOX039 TOX022

不純物FG*

不純物FH*

不純物FI*

不純物FJ*






Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

e 0.7%a 98.1% NAb NAb NAb 0.2%h

(RRT 0.83)

0.1% RRT 0.58 0.1% RRT 0.60 0.1% RRT 1.17 0.1% RRT 1.24 0.2% RRT 1.27d 0.1% RRT 1.63

0.9% TOX032

e,f 1.3%a 95.2% ND ND ND 0.21% 0.07% RRT 2.00 0.25% RRT 2.27c 0.09% RRT 2.30

0.05% RRT 2.42 0.05% RRT 2.47

0.63% TOX032

e,f 1.4%a 97.0% ND ND ND 0.29% 0.06% RRT 1.99 0.07% RRT 2.27 0.06% RRT 2.42

0.48% TOX032

不純物FG*

不純物FH*

不純物FI*

不純物FJ*






Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

e,f 1.8%a 97.1% ND ND ND 0.41% 0.05% RRT 0.74 0.05% RRT 1.45

0.05% RRT 1.50 0.07% RRT 1.52 0.10% RRT 1.99 0.15% RRT 2.27c 0.06% RRT 2.30

0.05% RRT 2.42 0.05% RRT 2.47

0.89% TOX032

f 1.1%a 98.0% ND ND ND 0.19% 0.10% RRT 2.27 0.05% RRT 2.30 0.05% RRT 2.42

0.29% TOX038 TOX103

0.5%a 99.0% ND ND ND 0.21% 0.06% RRT 0.67 0.05% RRT 1.45 0.05% RRT 1.50

0.28% TOX097

0.7% 99.6% ND ND ND 0.07% Trace 0.1% TOX153

不純物FG*

不純物FH*

不純物FI*

不純物FJ*



* Note: Footnotes are provided on the last page of the table. CONFIDENTIAL Page 51 17AUG2010



Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

0.3% 99.5% ND ND ND 0.1% 0.1% 0.2% TOX146

0.5% 99.8% ND ND ND 0.1% ND 0.1% TOX151 TOX152

NT 87.0 ND 0.07 ND ND 0.09% 0.35%

0.15% 0.88% 0.08% RRT 1.50

1.6 TX-162-2001

NT 100.0 ND ND ND 0.1 0.1% RRT 1.445 0.2 TOX108 TOX109

0.3 99.5 ND ND ND 0.1 0.1% 0.2 TOX108 TOX109

不純物FE*不純物FC*

不純物FF*不純物FK*

不純物FG*

不純物FH*

不純物FI*

不純物FJ*






Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

0.0 100.1 ND ND ND ND 0.17%

0.2 TX-164-2004

0.0 99.6 ND ND ND ND 0.05%

0.1 TX-164-2002; TX-164-2003

0.0 99.7 ND ND ND ND 0.10%

0.1 K01-3154 Note: material for K01-3154 was a mixture of

and

不純物FC*

不純物FC*

不純物FC*

不純物FG*

不純物FH*

不純物FI*

不純物FJ*






Total Impurities

(%)

(TP-

0274

)

Ass

ay b

y H

PLC

(w

/w)

(TP-

0228

) (T

P-03

52)

(T

P-03

53)

(TP-

0297

)


.5%

*

98.

0% &

102.

0%

0.1

0%

0.3

0%

0.1

0%

0.7

0%

0.1

0%

1.0

0%

Study Number

0.0 99.1 ND ND ND ND 0.12%

0.1 K01-3154 Note: material for K01-3154 was a mixture of

and

不純物FC*

不純物FG*

不純物FH*

不純物FI*

不純物FJ*


2.6.7.4.B. Toxicology Drug Substance Test Article: Rilpivirine



Batch No. Specified Impurities (%) PROPOSED

SPECIFICATION: Purity (%)

%

%

% Study No. Type of Study TMC278-Exp5463

Five-Day Repeated Dose Oral Toxicity Study in the Rat. / / / /

TMC278-Exp5461

Single Dose Escalation Oral Toxicity Study Followed by a 5-Day Repeated Dose Oral Toxicity Study in the Beagle Dog (tolerance study).

/ / / / TMC278-Exp5461

Single Dose Escalation Oral Toxicity Study Followed by a 5-Day Repeated Dose Oral Toxicity Study in the Beagle Dog (tolerance study).

/ / / / TMC278-Exp5627

Single Dose Oral Endocrinology Study in the Beagle Dog.

TMC278-NC136 2-Week Repeated Dose Oral Toxicity Study in the Rat. TMC278-NC117 1-Month Repeated Dose Oral Toxicity Study in the Rat. TMC278-NC116 1-Month Repeated Dose Oral Toxicity Study in the Beagle Dog. TMC278-NC101 6-Month Repeated Dose Oral Toxicity Study with 1-Month

Recovery in the Rat. TMC278-NC115 6-Month Repeated Dose Oral Toxicity Study with 3-Month

Interim Kill in the Beagle Dog. TMC278-NC126 5-Day Repeated Dose Oral Toxicity Study in the Female

Rabbit. TMC278-NC127 Pilot Oral Developmental Toxicity Study in the Rat. TMC278-NC105 Oral Developmental Toxicity Study in the Rat. TMC278-NC128 Pilot Oral Developmental Toxicity Study in the Rabbit. TMC278-NC129 Pilot Oral Developmental Toxicity Study in the Rabbit.

99.3 0.36 0.06 0.06

TMC278-NC130 Oral Developmental Toxicity Study in the Rabbit. 98.0 0.62 0.06 ND TMC278-NC107 52-Week Oral (Gavage) Toxicity Study in the Beagle Dog. 98.2 0.21 0.20 0.31 TMC278-NC279 In Vitro Bacterial Reverse Mutation Test of TMC278 in

Salmonella typhimurium with Human Liver S9-Mix. ND = not detected or <0.05 w/w%; / = not determined; is the free base of (HCI salt); = is the free base of (HCI salt);

= is the free base of (HCI salt)

不純物A* 不純物B'* 不純物C'*

不純物A* 不純物A'* 不純物B'* 不純物B'* 不純物B*不純物C'* 不純物C'* 不純物C*





SPECIFICATION: Purity

(%) %

%

% Study No. Type of Study TMC278-Exp5693

In Vitro Bacterial Reverse Mutation Test with Salmonella Typhimurium.

TMC278-Exp5692

4-Week Oral (gavage) Immunotoxicity Study in the Rat.

TMC278-Exp5650

1-Month Repeated Dose Oral Toxicity Study in the Beagle Dog with 1-Month Recovery.

98.5 0.61 0.14 0.16

TMC278-FK4790

A Study on the Effect of R278474 on the Biosynthesis of Cortisol in Crude Subcellular Fractions of Dog Adrenal Cortex.

TMC278-Exp5535

2-Week repeated Dose Oral Toxicity Study in the Rat.

TMC278-Exp5559

Single Dose Oral Toxicity Study in the Rat.

TMC278-Exp5534

7-Day Repeated Dose Oral Toxicity Study in the Beagle Dog.

TMC278-Exp5539

In Vitro Mammalian Forward Mutation Test with L5178Y Mouse Lymphoma Cells (TK-locus) using the Microtiter® Fluctuation Technique.

TMC278-Exp5540

In Vitro Bacterial Reverse Mutation Test with Salmonella Typhimurium.

TMC278-Exp5538

In Vivo Micronucleus Test on Bone Marrow Cells of Mice.

96.8 1.4 1.56 ND

TMC278-Exp5653

In Vitro Guinea Pig Adrenal Cell Test for the Evaluation of Cortisol Biosynthesis.

ND = not detected or <0.05 w/w%






SPECIFICATION Purity

(%) %

%

% Study No. Type of Study TMC278-NC118 2-Week Repeated Dose Oral Toxicity Study in the Swiss

Mouse. TMC278-NC136 2-Week Repeated Dose Oral Toxicity Study in the Rat. TMC278-NC177 2-Week Repeated Dose Oral Toxicity Study of R314585 in

the Rat. TMC278-NC117 1-Month Repeated Dose Oral Toxicity Study in the Rat. TMC278-NC116 1-Month Repeated Dose Oral Toxicity Study in the Beagle

Dog. TMC278-NC121 4-Week Toxicity Study by Oral Route (Gavage) in CB6F1-

nonTgrasH2 mice. TMC278-NC119 3-Month Repeated Dose Oral Toxicity Study in the Mouse. TMC278-NC125 Oral Female Fertility Study in the Rat. TMC278-NC168 Oral (Gavage) Pre and Post-natal Developmental Toxicity

and Juvenile Toxicity Dose Range Finding Study in the Rat.

100.3 0.37 0.09 0.05

TMC278-NC124 Oral (Gavage) Male Fertility Study in the Rat.

97.8 0.27 ND ND TMC278-NC124 Oral (Gavage) Male Fertility Study in the Rat. TMC278-NC131 Oral (Gavage) Pre and Post-Natal Developmental Toxicity

Study in the Rat. TMC278-NC188 In Vitro Phototoxicity Testing of R314585 by the Neutral

Red Uptake Assay Using Balb/c 3T3 Mouse Fibroblasts. TMC278-NC202 In Vitro Bovine Corneal Opacity-Permeability Eye Irritation

Test.

98.8 0.70 0.21 0.11

TMC278-NC199 Local Lymph Node Assay (LLNA). TMC278-NC159 Primary Skin Irritation Study in Rabbits (4-Hour Semi-

Occlusive Application). TMC278-NC120 Carcinogenicity Study by Oral Gavage Administration to

CD-1 Mice For 104 Weeks.

99.7 0.17 0.32 0.22

TMC278-NC123 Carcinogenicity Study by Oral Gavage Administration to CD Rats for 104 Weeks.







SPECIFICATION Purity

(%)

%

% % Study No. Type of Study TMC278-NC335 In Vitro Bacterial Reverse Mutation Test with R314585

(TMC278.HCl) in Salmonella typhimurium. TMC278-NC336 Mutation at the Thymidine Kinase (tk) Locus of Mouse

Lymphoma L5178Y Cells (MLA) Using the Microtitre Fluctuation Technique.

TMC278-NC311 Mutation at the Thymidine Kinase (tk) Locus of Mouse Lymphoma L5178Y (MLA) Using the Microtitre Fluctuation Technique.

TMC278-NC309 In Vitro Bacterial Reverse Mutation Test with R314585 (TMC278.HCl) spiked with 4% ( .HCl) in Salmonella typhimurium.

TMC278-NC308 In Vitro Bacterial Reverse Mutation Test with R314585 (TMC278.HCl) spiked with 4% ( .HCl) in Salmonella typhimurium.

TMC278-NC314 1-Month Repeated Dose Oral Impurity Qualification Study for TMC278.HCl in the Rat

99.6 0.18 0.12 ND

TMC278-NC312 Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) using the MicrotitreR fluctuation technique.

TMC278-NC120 Carcinogenicity Study by Oral Gavage Administration to CD-1 Mice For 104 Weeks.

98.8 0.42 0.28 0.14

TMC278-NC123 Carcinogenicity Study by Oral Gavage Administration to CD rats for 104 weeks.


98.6 0.88 0.23 0.15



100.4 ND 0.13 0.05


99.7 0.18 0.11 ND TMC278-NC248 Endocrinological Oral (gavage) 8-Week Study in the Female Sexually Immature Cynomolgus Monkey.



不純物C* 不純物C'*

不純物B'*不純物B*


2.6.7.4.C. Toxicology Drug Substance Test Article: Tenofovir disoproxil fumarate

* In initial batches was utilised. is now used. is also known as . = = . nd = none detected (LOD < 0.001%, LOQ = 0.005%) CONFIDENTIAL Page 58 17AUG2010


Purity (%) Organic volatile impurities (%)

Wat

er C

onte

nt (%

)

Enan

tiom

eric

Pur

ity

(%)

Ass

ay b

y H

PLC

(%)

Tot

al im

puri

ties b

y H

PLC

(%) (p

pm)

()

(

)

(%)

Con

tent

(ppm

)

SPECIFICATION: Batch No.

1.0

%

99.

0%

97.

0%

&

101

.0%

2.5

%

20

ppm

1.0

0%

0.2

0%

0.1

0%

0.1

0%

0.3

0%

98.

0%

&

102

.0%

I

nfo.

onl

y

Study Number Single Dose Toxicity

0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 R990200 0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 D990201

Repeat Dose Toxicity

0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 M990191 0.50 100.0 97.8 1.1 10 0.45 0.01 nd nd 0.01 100.8 65 M990203

0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 98-TOX-4331-004 0.33 99.5 96.0 2.9 10 1.57 0.11 nd 0.01 0.58 106.8 49 97-TOX-4331-002

0.45 99.4 96.3 2.1 10 0.44 0.01 nd nd 0.01 101.4 73 0.23 99.7 95.9 2.3 10 0.65 0.01 0.01 nd 0.04 100.1 54 97-TOX-4331-002 (cont) 0.39 98.7 97.0 2.0 10 0.31 0.49 0.01 nd 0.05 100.7 7



Purity (%) Organic volatile impurities (%) W

ater

Con

tent

(%)

Enan

tiom

eric

Pur

ity

(%)

Ass

ay b

y H

PLC

(%)

Tot

al im

puri

ties b

y H

PLC

(%) (p

pm)

()

(

)

(%)

Con

tent

(ppm

)


1.0

%

99.

0%

97.

0%

&

101

.0%

2.5

%

20

ppm

1.0

0%

0.2

0%

0.1

0%

0.1

0%

0.3

0%

98.

0%

&

102

.0%

I

nfo.

onl

y

Study Number

0.39 98.7 97.0 2.0 10 0.31 0.49 0.01 nd 0.05 100.7 7 98-TOX-4331-003 0.33 99.5 96.0 2.9 10 1.57 0.11 nd 0.01 0.58 106.8 49 97-TOX-4331-001

0.45 99.4 96.3 2.1 10 0.44 0.01 nd nd 0.01 101.4 73 0.23 99.7 95.9 2.3 10 0.65 0.01 0.01 nd 0.04 100.1 54 0.39 98.7 97.0 2.0 10 0.31 0.49 0.01 nd 0.05 100.7 7 0.4 100.0 98.8 0.5 <20 0.22 0.01 <0.01 nd 0.12 100.0 nt TX-164-2004

Genotoxicity 0.33 99.5 96.0 2.9 10 1.57 0.11 nd 0.01 0.58 106.8 49 97-TOX-1278-003 (

) 0.34 99.0 98.3 1.5 10 0.5 nd nd nd nd 101.2 133 97-TOX-4331-007

( )

0.34 99.0 98.3 1.5 10 0.5 nd nd nd nd 101.2 133 97-TOX-4331-008

0.3 99.0 0.7 0.37 0.01 0.35 23291-0-494OECD 0.3 100.0 100.1 0.7 <10 0.26 <0.01 0.01 nd 0.15 99.6 nt TX-164-2002; TX-164-2003

0.3 100.0 99.61 0.3 nt 0.17 <0.01 <0.01 nd 0.17 99.6 nt K01-3037




ater

Con

tent

(%)

Enan

tiom

eric

Pur

ity

(%)

Ass

ay b

y H

PLC

(%)

Tot

al im

puri

ties b

y H

PLC

(%) (p

pm)

()

(

)

(%)

Con

tent

(ppm

)


1.0

%

99.

0%

97.

0%

&

101

.0%

2.5

%

20

ppm

1.0

0%

0.2

0%

0.1

0%

0.1

0%

0.3

0%

98.

0%

&

102

.0%

I

nfo.

onl

y

Study Number Carcinogenicity

0.50 100.0 97.8 1.1 10 0.45 0.01 nd nd 0.01 100.8 65 M990205 0.50 100.0 97.8 1.1 10 0.45 0.01 nd nd 0.01 100.8 65 R990204

Reproduction Toxicity

0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 98-TOX-4331-006 (

) 0.34 99.0 98.3 1.5 10 0.5 nd nd nd nd 101.2 13

3 97-TOX-4331-004

0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 98-TOX-4331-005 0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 R990202

Local Tolerance 0.39 98.7 97.0 2.0 10 0.31 0.49 0.01 nd 0.05 100.7 7 B990165 0.39 98.7 97.0 2.0 10 0.31 0.49 0.01 nd 0.05 100.7 7 B990166




ater

Con

tent

(%)

Enan

tiom

eric

Pur

ity

(%)

Ass

ay b

y H

PLC

(%)

Tot

al im

puri

ties b

y H

PLC

(%) (p

pm)

()

(

)

(%)

Con

tent

(ppm

)


1.0

%

99.

0%

97.

0%

&

101

.0%

2.5

%

20

ppm

1.0

0%

0.2

0%

0.1

0%

0.1

0%

0.3

0%

98.

0%

&

102

.0%

I

nfo.

onl

y

Study Number Other Toxicity Studies

Antigenicity 0.39 98.7 97.0 2.0 10 0.31 0.49 0.01 nd 0.05 100.7 7 G990167

Mechanistic Studies

0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 R2000096 0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 R2000095 0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 R2000099 0.25 99.9 97.2 1.6 10 0.31 0.01 0.1 0.02 0.05 101.5 84 R2000043

0.50 100.0 97.8 1.1 10 0.45 <0.01 nd nd 0.01 100.8 65 R2000036 0.3 99.0 0.7 0.37 0.01 0.35 W2000042

0.50 100.0 97.8 1.1 10 0.45 <0.01 nd nd 0.01 100.8 65 P2000078

2.6.7.4.D. Toxicology Drug Substance (HPLC Impurity Profile) Test Article: Tenofovir disoproxil fumarate

Where no value is presented, the impurity was not detected (LOD 0.01%, LOQ= 0.05%), tr: <0.05%


2.6.7.4.D. Tenofovir disoproxil fumarate (HPLC Impurity Profile)

Specified Impurities (%)

Impurities by HPLC (%)

Impurity No. 3 4 6&7 8 9 10 10a 11 12 13 13a 14


1.0

0%

0.1

0%

0.3

0%

0.4

0%

0.6

0%

0.1

0%

0.1

0%

0.7

0%

0.6

0%

0.2

0%

0.1

0%

0.4

0%

Study Number Single Dose Toxicity

0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 R990200 0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 D990201

Repeat Dose Toxicity

0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 M990191 0.32 - - 0.17 0.12 0.03 0.05 0.39 0.42 - - 0.05 M990203

0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 98-TOX-4331-004 0.48 0.26 - 0.15 0.13 - 0.11 0.08 - 0.23 0.57 0.14 97-TOX-4331-002

0.68 0.02 - 0.16 0.17 - 0.03 0.13 - 0.19 0.45 0.17 0.24 0.06 - 0.11 0.15 - 0.12 - - 0.21 - 0.32 0.85 0.08 - 0.05 0.10 - 0.05 0.26 - 0.17 0.17 0.26

不純物TB*

不純物TN*

不純物TF*

不純物TG*

不純物TH*

不純物TP*

不純物TE*

不純物TI*

不純物TJ*

不純物TL*

不純物TO*

不純物TK*







Impurity No. 3 4 6&7 8 9 10 10a 11 12 13 13a 14


1.0

0%

0.1

0%

0.3

0%

0.4

0%

0.6

0%

0.1

0%

0.1

0%

0.7

0%

0.6

0%

0.2

0%

0.1

0%

0.4

0%

Study Number

0.85 0.08 - 0.05 0.10 - 0.05 0.26 - 0.17 0.17 0.26 98-TOX-4331-003 0.48 0.26 - 0.15 0.13 - 0.11 0.08 - 0.23 0.57 0.14 97-TOX-4331-001

0.68 0.02 - 0.16 0.17 - 0.03 0.13 - 0.19 0.45 0.17 0.24 0.06 - 0.11 0.15 - 0.12 - - 0.21 - 0.32 0.85 0.08 - 0.05 0.10 - 0.05 0.26 - 0.17 0.17 0.26 0.25 - - tr 0.20 - - - tr - - tr TX-164-2004

Genotoxicity 0.48 0.26 - 0.15 0.13 - 0.11 0.08 - 0.23 0.57 0.14 97-TOX-1278-003 (

) 0.26 - - 0.09 0.21 - - 0.16 - - 0.20 0.12 97-TOX-4331-007

( )

0.26 - - 0.09 0.21 - - 0.16 - - 0.20 0.12 97-TOX-4331-008

0.30 - - 0.12 0.19 - 0.05 - 0.07 - - - 23291-0-494OECD 0.58 - - - 0.12 - - - - - - tr TX-164-2002; TX-164-2003

0.20 - - - 0.10 - - - - - - - K01-3037

不純物TB*

不純物TN*

不純物TF*

不純物TG*

不純物TH*

不純物TP*

不純物TE*

不純物TI*

不純物TJ*

不純物TL*

不純物TO*

不純物TK*







Impurity No. 3 4 6&7 8 9 10 10a 11 12 13 13a 14


1.0

0%

0.1

0%

0.3

0%

0.4

0%

0.6

0%

0.1

0%

0.1

0%

0.7

0%

0.6

0%

0.2

0%

0.1

0%

0.4

0%

Study Number Carcinogenicity

0.32 - - 0.17 0.12 0.03 0.05 0.39 0.42 - - 0.05 M990205 0.32 - - 0.17 0.12 0.03 0.05 0.39 0.42 - - 0.05 R990204

Reproduction Toxicity

0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 98-TOX-4331-006 (

) 0.26 - - 0.09 0.21 - - 0.16 - - 0.20 0.12 97-TOX-4331-004

0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 98-TOX-4331-005 0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 R990202

Local Tolerance 0.85 0.08 - 0.05 0.10 - 0.05 0.26 - 0.17 0.17 0.26 B990165 0.85 0.08 - 0.05 0.10 - 0.05 0.26 - 0.17 0.17 0.26 B990166

不純物TB*

不純物TN*

不純物TF*

不純物TG*

不純物TH*

不純物TP*

不純物TE*

不純物TI*

不純物TJ*

不純物TL*

不純物TO*

不純物TK*







Impurity No. 3 4 6&7 8 9 10 10a 11 12 13 13a 14


1.0

0%

0.1

0%

0.3

0%

0.4

0%

0.6

0%

0.1

0%

0.1

0%

0.7

0%

0.6

0%

0.2

0%

0.1

0%

0.4

0%

Study Number Other Toxicity Studies

Antigenicity 0.85 0.08 - 0.05 0.10 - 0.05 0.26 - 0.17 0.17 0.26 G990167

Mechanistic Studies 0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 R2000096 0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 R2000095 0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 R2000099 0.55 0.02 0.27 0.12 0.09 0.04 0.04 0.20 - - 0.08 0.14 R2000043

0.32 - - 0.17 0.12 0.03 0.05 0.39 0.42 - - 0.05 R2000036 0.30 - - 0.12 0.19 - 0.05 - 0.07 - - - W2000042

0.32 - - 0.17 0.12 0.03 0.05 0.39 0.42 - - 0.05 P2000078 1.84 0.08 0.31 0.20 0.10 0.02 0.14 0.38 0.12 0.65 - 0.36 R2000081 0.89 - 0.22 0.22 0.11 - 0.06 0.37 0.13 - - 0.09

不純物TB*

不純物TN*

不純物TF*

不純物TG*

不純物TH*

不純物TP*

不純物TE*

不純物TI*

不純物TJ*

不純物TL*

不純物TO*

不純物TK*


2.6.7.4.E. Toxicology Drug Substance Test Article: Ground TDF/FTC Tablets

RRT = relative retention time CONFIDENTIAL Page 66 17AUG2010

2.6.7.4.E. Ground TDF/FTC Tablets

Strength by HPLC

Assay (%)

Impurity Content Tenofovir DF (Specified and Unspecified)

Impurity Content Emtricitabine (Specified and Unspecified)

Ten

ofov

ir D

F

Em

tric

itabi

ne

GS-

9039

GS-

9237

, ,

GS-

4921

27

,

SPECIFICATION Batch No.

90.

0% &

105

.0%

90.

0% &

1

05.0

%

6.0

%

5.0

0%

1.0

0%

1.0

0%

1.0

0%

0.7

5%

0.7

5%

0.5

0%

0.5

0%

0.2%

3.0

%

2.0

0%

1.0

0%

NA

NA

020

%.

Stud

y N

umbe

r:

( )

89.8 80.6 4.9 2.67 0.15 0.26 0.12 0.11 0.08 0.18 0.04 0.09 RRT 0.18 0.05 RRT 0.35 0.09 RRT 0.36 0.05 RRT 0.45 0.06 RRT 0.47 0.06

0.12 RRT 0.86

0.11

0.06 RRT 1.21

7.7 7.51 0.14 5.96 1.64 Dioxolane 0.04

TX-164-2001

( )

95.1 95.9 1.3 0.35 0.10 0.05 RRT 0.68 0.11

0.04 RRT 1.21

0.10 0.06 1.06 TX-164-2001

不純物TN*

不純物TO*

不純物TO*

不純物TB*

不純物TL*

不純物TM*

不純物TK*

不純物TD*

不純物TE*

不純物TR*

不純物TS*

不純物FE*

不純物FF''*

不純物FF'''*


2.6.7.4.F. Toxicology Drug Substance Test Article: Ground FTC/RPV/TDF Tablets


2.6.7.4.F. Ground FTC/RPV/TDF Tablets

Not applicable. No toxicology studies have been conducted with the FTC/RPV/TDF tablet.

2.6.7.5.A. Single-Dose Toxicity Test Article: Emtricitabine


2.6.7.5. Single-Dose Toxicity 2.6.7.5.A. Emtricitabine

Species/ Strain

Method of Administration (Vehicle/ Formulation)

Doses (mg/kg)

Gender and No.

per Group

Observed Maximum Nonlethal

Dose (mg/kg)

Approximate Lethal Dose

(mg/kg) Noteworthy Findings Study Number


Oral 4000 5 M 5 F

> 4000 4000 No clinical signs of toxicity were noted. No effects on body weight were noted. No gross pathology findings were noted.

TTEP/93/0020


Intravenous 200 5 M 5 F

> 200 200 No clinical signs of toxicity were observed. No effects on body weight were noted. No gross pathology findings were noted. TTEP/93/0023


Oral 4000 5 M 5 F

> 4000 4000 No clinical signs of toxicity were observed. No effects on body weight were noted. No gross pathology findings were noted.

TTEP/93/0021


Intravenous 200 5 M 5 F

> 200 200 No clinical signs of toxicity were observed. No effects on body weight were noted. No gross pathology findings were noted.

TTEP/93/0024

2.6.7.5.B. Single-Dose Toxicity Test Article: Rilpivirine

CA = citric acid, F = female; M = male; PEG400 = polyethylene glycol 400, RD = repeated dose; SD = single dose; TK: toxicokinetics; ND = not determined CONFIDENTIAL Page 69 17AUG2010


Species/ Strain

Route/Method of Administration (Vehicle/ Formulation) Batch no.

Doses (mg/kg)

Gender and No. per Group

NOAEL (mg/kg) Noteworthy Findings

Study Number

Rat/Sprague Dawley

Oral/Gavage (PEG400)

800 mg/kg 2M + 2F 4M + 4F (TK)

ND No mortality. Clinical signs: Salivation in all males and 2/6 females. Cmax = 18 (F) and 8.0 (M) g/mL and AUC0- = 233 (F) and 86 (M) g.h/mL.

TMC278-Exp5559

Dog/Beagle

Oral/Gavage

(PEG400) or (PEG400 + CA)

SD: 40 mg/kg (PEG400), 80 mg/kg (PEG400) 80 mg/kg (PEG400 + CA) RD (5-day): 80 mg/kg/day (PEG400 + CA)

1M + 1F/group

ND No mortality. SD/PEG 40 mg/kg: No effects Cmax = 1.1 (M) and 0.95 (F) g/mL and AUC0-24h = 21 (M) and 19 (F) g.h/mL. 80 mg/kg: Vomiting (1 and 2 days after dosing, male), soft feces (3 days after dosing (male) and 2 or 3 days after dosing (female)). Cmax = 0.88 (M) and 0.81 (F) g/mL and AUC0-24h = 17 (M) and 16 (F) g.h/mL SD/PEG +CA 80 mg/kg: No effects Cmax = 2.1 (M) and 2.2 (F) g/mL and AUC0-24h = 42 (M) and 49 (F) g.h/mL RD/PEG + CA 80 mg/kg: Salivation 4th day (male), vomiting 3rd day (male), gastrointestinal irritation. On day 5, Cmax = 13 (M) and 8.0 (F) g/mL and AUC0-24h = 262 (M) and 154 (F) g.h/mL.

TMC278-Exp5461


2.6.7.5.C. Single-Dose Toxicity Test Article: Tenofovir disoproxil fumarate 2.6.7.5.C. Tenofovir disoproxil fumarate

Species/ Strain


Doses (mg/kg)


Observed Maximum Nonlethal Dose (mg/kg)

Approximate Lethal Dose (mg/kg) Noteworthy Findings Study Number

Rat, Sprague Dawley

Oral Gavage

Suspension vehicle 0, 160, 500, 1500

5M

5F 1500 > 1500 NOAEL = 1500 mg/kg. R990200

Dog, Beagle

Oral Gavage

50mM citric acid solution.

0, 30, 90, 270

1M

1F

270 > 270

Renal tubular epithelial karyomegaly and basophilia, 90 & 270 mg/kg.

NOEL: 30 mg/kg. D990201

2.6.7.6.A. Repeat-Dose Toxicity Nonpivotal Studies Test Article: Rilpivirine


2.6.7.6. Repeat-Dose Toxicity: Nonpivotal Studies 2.6.7.6.A. Rilpivirine

Species/Strain No. and Gender/Group

Route/Method of Administration

(Vehicle/ Formulation) Duration Batch no.

Dosesa (mg/kg)


Study Number

Admixed to diet 0, 40, 400, 5000 mg/kg/day TMC278.HCl

/ 40 mg/kg: Cmax: 8.9-8.3 g/mL; AUC0-24h:150-148 g.h/mL (M-F) 400 mg/kg: Cmax: 57-63 g/mL; AUC0-24h: 1066-1320 g.h/mL (M-F) BWG (M: 22%, F: 17%), FC (M: 46%,

F: 39%), RBC (M: 8%), Hb (M: 7%), Hct (M: 8%), eosinophils (F: 48%), reticulocytes (M: 11%, F: 23%), bilirubin (M: 3-fold, F: 3-fold), ALP (M: 2-fold, F: 3-fold), AST (M: 1-fold, F: 3-fold), albumin (M: 13%), cholesterol (F: 22%), triglycerides (M: 50%, F: 73%). 5000 mg/kg: All animals died.

Mouse/Swiss Main animals: 5M/5F Satellite animals: 3M/3F

Oral gavage (0.5% w/v HPMC)

2-week

400, 2000 mg/kg/day TMC278.HCl

/ 400 mg/kg: BWG (M:13%, F: 29%), reticulocytes (M: 17%, F: 23%), bilirubin (F: 2-fold), ALP (M: 3-fold, F: 2-fold), ALT (M: 1-fold, F: 3-fold), AST (F: 2-fold), albumin (M: 22%), cholesterol (F: 48%), calcium (M: 8%, F: 5%), protein (M: 13%), urea (M: 19%, F: 40%), triglycerides (F: 63%). Cmax: 63-94 g/mL; AUC0 24h: 687 123 g.h/mL (M-F) 2000 mg/kg: All animals died.

TMC278-NC118

a dose of HCl salt is presented as base equivalents; ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; BWG: body weight gain; F: female; FC: food consumption; Hb: hemoglobin; Hct: hematocrit. HPMC: hydroxypropyl methyl cellulose; M: male; RBC: Red blood cells; WBC: white blood cells; / = not determined.






Dosesa (mg/kg)


Study Number

Mouse/CB6F1-nonTgrasH2 Main animals: 10M/10F Satellite animals: 15M/15F

Oral/gavage (0.5% w/v HPMC)

1-month

0, 20, 80, 320 mg/kg/day TMC278.HCl

/ 20 mg/kg: Liver: hypertrophy, Uterus: atrophy Cmax: 14-15 g/mL; AUC0-24h: 64-54 g.h/mL (M-F) 80 mg/kg: liver (M: 24%, F: 29%). Liver: hypertrophy, Kidney: degenerative/necrotic nephropathy. Cmax: 30-37 g/mL; AUC0-24h: 250 240 g.h/mL (M-F) 320 mg/kg: 2 mortalities. BW and FC. RBC (M: 4%), Hb (M: 4%, F: 4%), Hct (F: 6%), reticulocytes (F: 56%), platelets (M: 14%, F: 24%), calcium (F: 72%), IP (F: 42%), bilirubin (F: 3-fold), urea (M: 72%, F: 58%), protein (M:9%, F: 18%), albumin (M: 17%), cholesterol (M: 46%, F: 110%), ALP (M: 3-fold,F: 2-fold). liver (M: 2-fold, F: 2-fold), kidney (F: 14%), spleen (F: 40%), ovaries (50%), thymus (F: 2!%). Liver: hypertrophy, Kidney: degenerative/necrotic nephropathy, Thymus: lymphoid depletion, Bone marrow: myeloid cell hyperplasia, Spleen: erythro/myelopoiesis, Uterus: atrophy. Cmax: 66 69 g/mL; AUC0-24h: 1090 942 g.h/mL (M-F)

TMC278-NC121b

Rat/Sprague Dawley 5M

Oral/gavage (PEG400)

5-day

0 (water), 0 (vehicle), 40, 400 mg/kg/day TMC278 base

40 (NOEL)

40 mg/kg: No effects Cmax: 1.4 g/mL; AUC0-24h: 14 g.h/mL 400 mg/kg: potassium (7%), No histopathology changes Cmax: 1.8 g/mL; AUC0-24h: 28 g.h/mL

TMC278-TOX5463

a dose of HCl salt is presented as base equivalents; b: GLP compliant study; ALP: alkaline phosphatase; AUC0 24h : area under the concentration vs time curve untill 24 hours after dosing; BW: body weight; Cmax: maximum concentration; F: female; FC: food consumption; Hb: hemoglobin; Hct: hematocrit. HPMC: hydroxypropyl methyl cellulose; IP: inorganic phosphate; M: male; RBC: red blood cells; WBC: white blood cells; / = not determined






Doses a (mg/kg)


Study Number

Rat/Sprague Dawley Main groups: 5M/5F Satellite groups: 4M/4F

Oral/gavage (PEG400 + CA)

14-day

0 (water), 0 (vehicle), 40, 120, 400 mg/kg/day TMC278 base

/ 40 mg/kg: T4 (M: 10%, F: 32%). Thyroid: hypertrophy (M). Cmax = 2.2-5.7 g/mL (M-F). AUC0-24h = 16-42 g.h/mL (M-F) 120 mg/kg: TSH (M: 94%, F: 40%); T4 (M: 27%, F: 50%). Thyroid: hypertrophy Cmax = 3.6-7.8 g/mL (M-F); AUC0-24h = 35-88 g.h/mL (M-F) 400 mg/kg: protein (M: 3%); albumin (M: 7%). TSH (M: 120%, F: 51%); T4 (M: 29%,

F: 66%). thyroid weight (36%, F).

Thyroid: hypertrophy. Cmax = 8.4-15 g/mL (M-F); AUC0-24h = 84-152 g.h/mL (M-F)

TMC278-TOX5535b

a dose of HCl salt is presented as base equivalents; b: GLP compliant study; ALT: alanine aminotransferase; AUC0-24h : area under the concentration vs time curve till 24 h after dosing; CA: citric acid; Cmax: maximum concentration; F: female; Hb: hemoglobin; Hct: hematocrit. HPMC: hydroxypropyl methyl cellulose, M: male; PEG400: polyethylene glycol 400; T4: tetraiodothyronine/thyroxine; TSH: thyroid stimulating hormone; WBC: white blood cells; / = not determined.






Doses a (mg/kg)


Study Number

Admixed to diet

and

0 (control diet), 400, 1200 mg/kg/day TMC278.HCl or 400 mg/kg/day TMC278 base

/ 400 mg/kg (TMC278.HCl-diet): WBC (F: 13%); ALT (M: 33%); thyroid weight (M: 20%, F: 54%). Thyroid: hypertrophy Cmax: 5.2-7.2 g/mL; AUC0-24 h= 82-137 g.h/mL (M-F). 400 mg/kg (TMC278 base-diet): WBC (M: 18%, F: 23%); Hb (M: 6%); Hct (M: 6%); thyroid weight (M: 27%, F: 39%).

Thyroid: hypertrophy Cmax: 3.7-4.3 g/mL; AUC0-24 h= 57-86 g.h/mL (M-F) 1200 mg/kg (TMC278.HCl-diet): WBC (F: 9%); Hb (M: 6%); Hct (M: 6%); ALT (M: 44%); liver weight (M: 30%, F: 18%); thyroid weight (M: 60%, F: 77%). Liver:

hypertrophy, Thyroid: hypertrophy. Cmax: 11-13 g/mL; AUC0-24h = 180-266 g.h/mL (M-F)

Rat/Sprague Dawley Main groups: 5M/5F Satellite groups:6M/6F

Oral/Gavage (0.5% w/v HPMC)

14-day

400 mg/kg/day TMC278.HCl

400 mg/kg (TMC278.HCl-gavage): WBC (F: 19%); cholesterol (M: 29%); ALT (M: 19%); thyroid weight (M: 33%, F: 39%). Thyroid: hypertrophy Cmax: 7.3-12 g/mL; AUC0-24h = 51-103 g.h/mL (M-F)

TMC278-NC136

a dose of HCl salt is presented as base equivalents; ALT: alanine aminotransferase; AUC0-24h : area under the concentration vs time curve till 24 h after dosing; CA: citric acid; Cmax: maximum concentration; F: female; Hb: hemoglobin; Hct: hematocrit. HPMC: hydroxypropyl methyl cellulose, M: male; PEG400: polyethylene glycol 400; T4: tetraiodothyronine/thyroxine; TSH: thyroid stimulating hormone; WBC: white blood cells; / = not determined.






Doses a (mg/kg)


Study Number



2-week

0 (vehicle), 400, 1500, 2000 mg/kg/day TMC278.HCl

/ 400 mg/kg: thyroid weight (F: 42%). Thyroid: hypertrophy (F). Cmax = 5.2-12 g/mL (M-F); AUC0-24h= 42-96 g.h/mL (M-F) 1500 mg/kg: APTT (M: 19%), PT (M: 20%); chloride (F: 3%); glucose (F: 18%); urea nitrogen (F: 33%); creatinine (F: 23%). thyroid weight (F: 62%). Thyroid:

hypertrophy. Cmax = 9.5-12 g/mL (M-F); AUC0-24h= 86-115 g.h/mL (M-F) 2000 mg/kg: APTT (M: 14%), PT (M: 15%); chloride (F: 3%); cholesterol (M: 30%); glucose (F: 14%). urea nitrogen (F: 33%); creatinine (F: 17%). thyroid weight (F: 55%). Thyroid:

hypertrophy. Cmax = 7.7-14 g/mL (M-F); AUC0-24h= 77-147 g.h/mL (M-F)

TMC278-NC177

a dose of HCl salt is presented as base equivalents; ALT: alanine aminotransferase; APTT: activated partial thromboplastine time; AUC0-24h : area under the concentration vs time curve till 24 h after dosing; Cmax: maximum concentration; F: female; HPMC: hydroxypropyl methyl cellulose, M: male; PT: Prothrombin time; T4: tetraiodothyronine/thyroxine; TSH: thyroid stimulating hormone; WBC: white blood cells; / = not determined.






Dosesa (mg/kg)


Study Number


0 (vehicle), 10, 400 mg/kg/day TMC278 base

/ 10 mg/kg: Thyroid: hypertrophy. Cmax = 0.74-1.4 g/mL (M-F); AUC0-24h = 5.4-8.6 g.h/mL (M-F) 400 mg/kg: Hct (3%); potassium (13%); chloride (F: 2%); cholesterol F: (22%); urea nitrogen (F: 15%); albumin (M: 7%); protein (M: 5%); TSH (M: 51%, F: 15%); T3 (M: 57%);

T4.(M: 61%, F: 52%). liver weight (M: 24%, F: 15%); thyroid

weight (F: 60%). Liver: hypertrophy, Thyroid: hypertrophy. Cmax = 7.7-13 g/mL (M-F); AUC0-24h = 90-149 g.h/mL (M-F)



1-month

0 (vehicle), 10, 400 mg/kg/day TMC278.HCl

/ 10 mg/kg: Thyroid: hypertrophy Cmax = 0.76-1.7 g/mL (M-F); AUC0-24h= 4.5-7.8 g.h/mL (M-F) 400 mg/kg: RBC (F: 4%); albumin (M: 5%); cholesterol (F: 10%); triglycerides (F: 35%); urea nitrogen (M: 18%, F: 11%); TSH (M: 49%, F: 38%), T3 (M: 56%); T4 (M: 37%, F: 44%); thyroid weight (F: 29%). Liver: hypertrophy, Thyroid: hypertrophy Cmax = 4.8-9.0 g/mL (M-F); AUC0-24h= 33-86 g.h/mL (M-F)

TMC278-NC117b

a dose of HCl salt is presented as base equivalents; b: GLP compliant study; AUC0-24h : area under the concentration vs time curve till 24 h after dosing; CA: citric acid; Cmax: maximum concentration; F: female; Hct: hematocrit; HPMC: hydroxypropyl methyl cellulose; M: male; PEG400: polyethylene glycol 400; RBC: red blood cells; T3: triiodothyronine; T4: tetraiodothyronine, thyroxine; TSH: thyroid stimulating hormone; / = not determined.






Doses (mg/kg)


Study Number

Rabbit/Albino New Zealand White 5F/group

Oral/gavage (0.5% HPMC)

5-day 0 (vehicle), 100, 300, 1000 mg/kg/day TMC278 base

/ 100 mg/kg: body weight (8%), food consumption (23%). Cmax: 57.3 g/mL; AUC0-24h: 1120

g.h/mL 300 mg/kg: body weight (14%), food consumption (62%). reticulocytes (61%); creatinine (51%).

Cmax: 124 g/mL; AUC0-24h: 2695 g.h/mL 1000 mg/kg: body weight (22%), food consumption (92%). reticulocytes (78%); creatinine (63%).

Cmax: 138 g/mL; AUC0-24h: 2971 g.h/mL

TMC278-NC126

AUC0-24h : area under the concentration vs time curve till 24 h after dosing; Cmax: maximum concentration; F: female; HPMC: hydroxypropyl methyl cellulose; / = not determined.






Doses (mg/kg)


Study Number

Dog/Beagle 4M/group

Oral/gavage PEG400 + CA

7-day

0 (water), 0 (vehicle), 20, 40, 80 mg/kg/day TMC278 base

/ 0 (water): Vomiting, salivation, soft feces. 0 (vehicle): Vomiting, salivation, soft feces. Focal congestion in the small intestine. 20 mg/kg: Vomiting, salivation, soft feces. cortisol (44%); Small intestines: Focal

congestion and mucosal hemorrhages. Adrenal glands: Multifocal inflammatory cell infiltration and multifocal vacuolated cells in the zona fasciculata Cmax: 3.7 g/mL; AUC0-24h: 39 g.h/mL 40 mg/kg: Vomiting, salivation, soft feces. bilirubin (2-fold), glucose (7%) and

cholesterol (17%); cortisol (72%); Small intestines: Focal congestion and mucosal hemorrhages. Adrenal glands: Multifocal inflammatory cell infiltration and multifocal vacuolated cells in the zona fasciculata. Cmax: 7.7 g/mL; AUC0-24h: 159 g.h/mL 80 mg/kg: Vomiting, salivation, soft feces. glucose (10%), cholesterol (12%) and

bilirubin (2-fold); cortisol (56%); Small intestines: Focal congestion and mucosal hemorrhages. Adrenal glands: Multifocal inflammatory cell infiltration and multifocal vacuolated cells in the zona fasciculata. Cmax: 7.8 g/mL; AUC0-24h: 147 g.h/mL

TMC278-TOX5534

AUC0-24h: area under the concentration vs time curve till 24 h after dosing; CA: citric acid; Cmax: maximum concentration; M: male; PEG400: polyethylene glycol 400; / = not determined.





(Vehicle/Formulation) Duration Batch no. Doses

(mg/kg) NOAEL (mg/kg) Noteworthy Findings

Study Number

Dog/Beagle 3M/3F


1-month 0 (vehicle), 5, 40 mg/kg/day TMC278 base

/ 5 mg/kg: Uterus: increased glandular development. Cmax = 1.4-1.1 g/mL (M-F); AUC0-24h = 23-14 g.h/mL (M-F) 40 mg/kg: RBC (F: 7%, F), Hct (M: 12%, F: 5%), reticulocytes (M: 61%).

IP (F: 11%), triglycerides (M: 53%), bilirubin (M: 39%, F: 62%), ALP (M:

1-fold, F: 2-fold), ALT (M: 5-fold, F: 2-fold), GT (M: 3-fold); ACTH (M: 77%, F: 92%), progesterone (M: 10-fold), 17 -OH-progesterone (M: 34-fold), cortisol (M: 47%, F: 34%); Liver: Centrilobular and periportal inflammation, brown pigmented macrophages, single cell death and oval cell proliferation. Adrenal glands: Swollen cells with densely stained cytoplasm in zona reticularis and fasciculata and presence of foamy cells in the zona fasciculata. Ovaries: in cystic luteinized follicles or tertiary follicles. Uterus: in glandular development. Testes: Prominent Leydig cells. Cmax = 3.8-2.5 g/mL (M-F); AUC0-24h= 73-43 g.h/mL (M-F)

TMC278-NC116a

a GLP compliance; ACTH: adrenocorticotropic hormone; ALT: alanine aminotransferase, ALP: alkaline phosphatase; AUC0-24h : area under the concentration vs time curve till 24 h after dosing; CA: citric acid; Cmax: maximum concentration; F: female; GT: gamma glutamyltransferase; Hct: hematocrit; IP: inorganic phosphate; M: male; PEG400: polyethylene glycol 400; RBC: red blood cell; / = not determined.





(Vehicle/Formulation) Duration Batch no. Dosesa

(mg/kg) NOAEL (mg/kg) Noteworthy Findings

Study Number

Dog/Beagle 3M/3F


1-month 0 (vehicle), 5, 40 mg/kg/day TMC278.HCl

/ 5 mg/kg: IP (F: 10%). Uterus: in glandular development Cmax = 0.89-0.70 g/mL (M-F); AUC0-24h = 13-7.5 g.h/mL (M-F) 40 mg/kg: Hct (M: 15%, F: 7%), reticulocytes (M: 43%). IP (F: 8%), bilirubin (M: 17%, F:100%),

ALP (M: 3-fold, F: 3-fold), AST (F: 1-fold), ALT (M: 1-fold, F: 6-fold), GT (F: 6-fold), ACTH (M: 59%, F: 54%), cortisol (M: 54%), progesterone (M: 10-fold), 17 -OH-progesterone (M: 29-fold). Liver: Centrilobular and periportal inflammation, brown pigmented macrophages, single cell death and oval cell proliferation. Adrenal glands: Swollen cells with densely stained cytoplasm in zona reticularis and fasciculata and presence of foamy cells in the zona fasciculata. Ovaries: in cystic luteinized follicles or tertiary follicles. Uterus: in glandular development. Testes:

Prominent Leydig cells. Cmax = 4.1-5.8 g/mL (M-F); AUC0-24h = 76-81 g.h/mL (M-F)

TMC278-NC116b

a dose of HCl salt is presented as base equivalents; b: GLP compliant study; ACTH: adrenocorticotropic hormone; ALT: alanine aminotransferase, ALP: alkaline phosphatase; AST: aspartate aminotransferase; AUC0-24h : area under the concentration vs time curve till 24 h after dosing; Cmax: maximum concentration; F: female; GT: gamma glutamyltransferase; Hct: hematocrit; HPMC: hydroxypropyl methyl cellulose; M: male; / = not determined.

2.6.7.6.B. Repeat-Dose Toxicity Nonpivotal Studies Test Article: Tenofovir disoproxil fumarate


2.6.7.6.B. Tenofovir disoproxil fumarate

Species/ Strain


Duration of Dosing

Doses (mg/kg)


NOAELa

/NOELb

(mg/kg) Noteworthy Findings Study Number

Mouse, ICR CD-1

Oral gavage 13 days 0, 100, 300, 1000

5 M & 5 F 100 mg/kgb Marked of platelet counts in 2/8 F at 1000 mg/kg. Dose-related in ALT activity;

ALP activity and phosphorus concentration at 300 and 1000 mg/kg. Minimal in incidence of single cell necrosis and centrilobular hypertrophy in the liver and minimal renal proximal tubular epithelial karyomegaly in the 1000 mg/kg group.

M990191

Rat, Sprague Dawley

Oral gavage 5 days 0, 25, 100, 400

4 M 100 mg/kg 400 mg/kgb

Slight in terminal (Day 8) mean body weights at 400 mg/kg.

96-TOX-4331-002

Dog, Beagle


2 M 9 mg/kgb NOEL: 9 mg/kg/d Non-formed, mucoid, liquid or no feces, emesis, and body wgts and food consumption at 45 and 180 mg/kg/d. Mild to marked BUN and creatinine (45 mg/kg/d also), marked phosphorus and potassium, and marked calcium and chloride at 180 mg/kg/d. Renal tubular cell degeneration/regeneration and cell necrosis were noted at 45 and 180 mg/kg/d. Bone marrow hypocellularity, lymphocytic depletion of spleen, gut associated lymphoid tissues, and lymph nodes, and cryptal epithelial cell necrosis of small and large intestines were noted at 180 mg/kg/d.

96-TOX-4331-001

a No Observed Adverse Effect Level; b No Observed Effect Level

2.6.7.7.A. Repeat-Dose Toxicity Report Title: 14 Day Oral (Gavage) Toxicity Study in Mice Given FTC

Test Article: Emtricitabine


2.6.7.7. Repeat-Dose Toxicity: Pivotal Studies 2.6.7.7.A. IUW00701: 14 Day Oral (Gavage) Toxicity Study in Mice Given FTC

Species/Strain: Mouse / Charles River CD-1 Duration of Dosing: 14 days Study Number: IUW00701 Initial Age: Approximately 3-4 weeks old Duration of Postdose: 14 days Date of First Dose: 19 Method of Administration: Oral, divided doses spaced

by approximately six hours

Vehicle/Formulation: Vehicle control: 0.5% methylcellulose

GLP Compliance: Yes

No Observed Adverse-Effect Level: 3000 mg/kg

Daily Dose (mg/kg) 0 (Control) 120 600 3000 Number of Animals M: 15 F: 15 M: 15 F: 15 M: 15 F: 15 M: 15 F: 15 Toxicokinetics: Plasma Conc. ( g/mL; Mean ± SD) Day 2 N/A 12.2 ± 1.87 11.7 ± 1.39 62.7 ± 9.69 63.0 ± 2.83 318 ± 4.3 358 ± 108.2

Day 14 N/A 13.6 ± 3.37 14.7 ± 10.01 66.9 ± 2.48 55.4 ± 2.04 389 ± 85.4 405 ± 59.2 Noteworthy Findings

Died or Sacrified MoribundA 1 0 0 0 0 0 0 1 Body Weight (%) There were no findings Food consumption (%) There were no findings Water consumption Not measured Clinical Observations There were no findings in either sex which were considered to be due to treatment with emtricitabine. Ophthalmoscopy There were no findings. All animals were examined before the start of the study and all animals from groups 1 and 4

(controls and high dose) were examined during week 2 of treatment. Ophthalmoscopic examinations were performed using an indirect and a direct ophthalmoscope and a mydriatic agent (1% tropicamide) to aid examination.

Electrocardiography Not measured

A Two mice died during the study, one male in the control group and a female in the 3000 mg/kg/day group. Both were considered to be a result of the dosing technique (oral gavage).

2.6.7.7.A. Repeat-Dose Toxicity Report Title: 14 Day Oral (Gavage) Toxicity Study in Mice Given FTC



Daily Dose (mg/kg) 0 (Control) 120 600 3000 Number of Animals M: 15 F: 15 M: 15 F: 15 M: 15 F: 15 M: 15 F: 15 Hematology There were no findings Serum Chemistry There were no findings Urinalysis Not performed Organ Weights (%) There were no findings Gross Pathology Necropsy There were no findings Histopathology There were no findings Additional Examinations Proof of Absorption – An additional 3 groups of 8 male and 8 female CD-1 mice received 120, 600, and

3000 mg/kg/day emtricitabine, by oral gavage, daily, split over two doses, for 2 or 14 days, to provide blood samples for proof of absorption. Blood samples were obtained from three males and three females from these groups approximately 45 minutes after the first dose on that day. Additional 5 male and 5 female mice were assigned to groups 1-4 to give blood for a pre-study blood screen.

Postdose Evaluation: At the end of the dosing period, 5 males and 5 females from each group continued without dosing for 14 days to investigate recovery.

2.6.7.7.B. Repeat-Dose Toxicity Report Title: A Thirty-Day Oral Toxicity Study in Mice Given 524W91



2.6.7.7.B. TOX599: A Thirty-Day Oral Toxicity Study in Mice Given 524W91

Species/Strain: Mouse / Charles River CD-1 Duration of Dosing: 30-32 days Study Number: TOX599 Initial Age: Approximately 48 day males and 52 day females

Duration of Postdose: 13 days for post-dose recovery

Date of First Dose: 19 Method of Administration: Oral Vehicle/Formulation: Vehicle control: 0.5%

aqueous methylcellulose GLP Compliance: Yes

No Observed Adverse-Effect Level: 600 mg/kg Daily Dose (mg/kg) 0 (Control) 120 600 3000 Number of Animals M: 14 F: 14 M: 14 F: 14 M: 14 F: 14 M: 14 F: 14 Toxicokinetics:

AUCssA( g.h/mL) Day 3 N/A N/A 78.1 83.1 410 326 1800 1711 Day 32 N/A N/A 103 73.9 499 304 1680 1320

Cmax ss ( g/mL) Day 3 N/A N/A 40.8 52.2 200 196 749 623 Day 32 N/A N/A 52.2 38.8 219 211 742 675

Tmax (hr) Day 3 N/A N/A 0.5 0.5 0.5 0.5 0.5 0.5 Day 32 N/A N/A 0.5 0.5 0.5 0.5 1.0 1.0

Cl / f (L/kg/h) Day 3 N/A N/A 1.54 1.45 1.46 1.84 1.67 1.75 Day 32 N/A N/A 1.17 1.63 1.2 1.97 1.55 1.98

Noteworthy Findings Died or Sacrified MoribundB 0 0 0 1 0 0 4 2 Body Weight (%) No treatment related changes Food Consumption (%) No treatment related changes Water Consumption Not measured Clinical Observations No treatment related changes Ophthalmoscopy No treatment related changes Electrocardiography Not measured

A Drug plasma concentrations – Cmax and AUC increased with dose and values were similar between males and females. Cmax and AUC were comparable at day 3 and day 32, indicating no signs of drug accumulation with repeated dosing.

B Seven animals died, one animal in the 120 mg/kg/day group and 6 in the 3000 mg/kg/day group. Cause of death was either mis-dosing, or probable mis-dosing in 5 animals. The cause of death was undetermined in 2 high-dose mice (3000 mg/kg/day), and a relationship to drug treatment could not be ruled out.

2.6.7.7.B. Repeat-Dose Toxicity Report Title: A Thirty-Day Oral Toxicity Study in Mice Given 524W91



Daily Dose (mg/kg) 0 (Control) 120 600 3000 Number of Animals M: 14 F: 14 M: 14 F: 14 M: 14 F: 14 M: 14 F: 14 Hematology The once-daily administration of 3000 mg/kg for 30–32 days resulted in decreased total erythrocyte count, hemoglobin, and

hematocrit and increased mean corpuscular volume, mean corpuscular hemoglobin, and red cell distribution width. The no-effect level for hematological changes was 600 mg/kg/day.

Hematocrit D* t Hemoglobin D t Total erythrocyte count D* D* t Mean corpuscular volume I* I t Mean corpuscular hemoglobin I* I* t Red cell distribution width I* I* t

Serum Chemistry No treatment related changes Urinalysis No treatment related changes Organ Weights (%) In females, mean relative organ weight (for pituitary, heart, spleen, ovary) increased at 3000 mg/kg/day. In males, mean relative

spleen weight increased at 3000 mg/kg/day and decreased for testes and thymus at 3000 mg/kg/day. The weight changes were unaccompanied by any histopathological alterations.

Changes in organ weights D-Thymus I-Pituitary (no histopathological changes) D-Testes I-Heart

I-Spleen I-Ovary I-Spleen

Gross Pathology No treatment related changes HistopathologyC No treatment related changes Additional Examinations - Plasma drug levels were determined for groups 5-7 at 0.5, 1, 3, 6, 12, and 24 hours after dosing on days 3 and 32.

Group 5: 120 mg/kg/day – 38 male; 38 female Group 6: 600 mg/kg/day – 38 male; 38 female Group 7: 3000 mg/kg/day – 38 male; 38 female - Ophthalmic examination (pupillary dilation was achieved with 1% tropicamide ophthalmic solution). - Heart and skeletal muscle examined by electron microscopy.

Postdose Evaluation: Four mice/sex/group held 13 days for post-dose recovery.

C Electronmicroscopy – No drug related ultrastructural changes noted in heart and skeletal muscle sampled from 5 mice/sex/group and from 5 males and 5 females from the control and 3000 mg/kg/day groups.

D = decrease I = increase p = permanent t = transitory * = p 0.05 or 0.01

2.6.7.7.C. Repeat-Dose Toxicity Report Title: Six Month Oral (Gavage) Toxicity Study in Mice Given FTC with a 3-Month Interim Kill



2.6.7.7.C. TOX022: Six Month Oral (Gavage) Toxicity Study in Mice Given FTC with a 3-Month Interim Kill

Species/Strain: Mouse / Charles River CD-1 Duration of Dosing: 13 weeks – 10 mice/sex/dose 26 weeks – 15 mice/sex/dose

Study Number: TOX022

Initial Age: 4 – 5 weeks Duration of Postdose: 1 month Date of First Dose: 19 Method of Administration: Oral Vehicle/Formulation: Vehicle control: 0.5%


No Observed Adverse-Effect Level: No observed effect level (NOEL) – 500 mg/kg/day after administration for 6 months.

Daily Dose (mg/kg) 0 (Control) 167 500 1500 Number of Animals M: 30 F: 30 M: 30 F: 30 M: 30 F: 30 M: 30 F: 30 Toxicokinetics:

AUC 0-24 ( g.h/mL) 3 months N/A N/A 63.8 67.9 181.4 209.6 513.4 569.5 6 months N/A N/A 82.3 93.1 248.4 284.4 732.1 899.4

Cmax ( g/mL) 3 months N/A N/A 44.0 37.2 128.0 88.8 225.0 187.1 6 months N/A N/A 41.7 48.6 87.3 111.7 310.9 306.0

T1/2 (hr) 3 months N/A N/A 4.71 3.25 2.51 3.00 2.86 4.20 6 months N/A N/A 2.95 3.01 3.05 3.91 2.67 3.18

A Satellite groups 40 males, 40 females. Dosed for 13 or 26 weeks, whereupon 3 animals/group/sex were bled at 6 time points for toxicokinetic samples. Evaluations were made after 3 months and 6 months of dosing, at 0 (pre-dose), 0.5, 1, 2, 5 and 8 hours after dosing. Tmax was 30-60 minutes after dosing; AUC0-24 and Cmax at steady state increased linearly with increased dose; t1/2 3-4 hours; there were no apparent differences in t1/2 estimates between Week 13 and Week 26; AUC and Cmax estimates in both sex groups at Week 26 were higher than at Week 13 especially for the highest dose level (possibly due to inter-animal variabilities or reduced kidney function in the older mice); there were no significant differences in plasma exposure or t1/2 between male and female mice.

2.6.7.7.C. Repeat-Dose Toxicity

Report Title: Six Month Oral (Gavage) Toxicity Study in Mouse Given FTC with a 3-Month Interim Kill



Daily Dose (mg/kg) 0 (Control) 167 500 1500 Number of Animals M: 30 F: 30 M: 30 F: 30 M: 30 F: 30 M: 30 F: 30 Noteworthy Findings

Died or Sacrified Moribund 2 0 0 0 0 0 0 0 There were 4 deaths during the study. One control male was killed on day 7 for humane reasons (had shown

convulsions, piloerection, and unsteady gait on day 6; no notable findings at necropsy) and a further control male was killed on day 128 for humane reasons (animal was prostrate, subdued, had weight loss, and had lost the use of the hindlimbs; no notable findings at necropsy but at histopathological evaluation had evidence of a possible spinal injury). A group 6 male was found dead (following a convulsion and also showed weight loss) on day 85 and 1 group 7 male was found dead on day 25 (no clinical signs). Deaths were considered unrelated to emtricitabine treatment.

Body Weight (%) Not reported Food consumption (%) There was a statistically significant reduction in food consumption in males in all emtricitabine-treated groups (167,

500 and 1500 mg/kg/day) at a single time-point (week 22–23 only). There was no statistically significant difference in food consumption compared to controls at week 18 or week 26 during the dosing period.

Water consumption Not measured Clinical Observations No treatment related changes Ophthalmoscopy No treatment related changes Electrocardiography Not measured Hematology Mean corpuscular volume and mean cell hemoglobin showed a small increase at 1500 mg/kg/day at week 27. This was

more notable in females and was still slightly elevated in females at the end of the recovery period. Serum Chemistry No treatment related changes Urinalysis Not measured Organ Weights (%) No treatment related changes Gross Pathology No treatment related changes Histopathology No treatment related changes Additional Examinations None Postdose Evaluation: 1 month after 26 w dosing - 5 mice/sex/dose

2.6.7.7.D. Repeat-Dose Toxicity

Report Title: A Six Month Oral Toxicity Study (With a Three Month Interim Sacrifice) in Mice Given 524W91



2.6.7.7.D. TOX628: A Six Month Oral Toxicity Study (With a Three Month Interim Sacrifice) in Mice Given 524W91

Species/Strain: Mouse / Charles River CD-1 Duration of Dosing: 6m (plus 3m interim kill) Study Number: TOX628 Initial Age: Approx. M: 62 days; F: 67 days Duration of Postdose: 3 weeks Date of First Dose: 19 Method of Administration: Oral Vehicle/Formulation: Vehicle control: 0.5%


No Observed Adverse-Effect Level: No effect level (NOEL) – 600 mg/kg/day


AUCA ( g.h/mL) N/A N/A 170 115 509 427 2026 1549 Cmax ( g/mL) N/A N/A 69.0 44.8 152 188 635 503

Noteworthy Findings Died or Sacrified MoribundB 1 0 0 0 0 0 1 1 Body Weight (%) No treatment related changes Food Consumption (%) No treatment related changes Water Consumption Not measured Clinical Observations No treatment related changes Ophthalmoscopy No treatment related changes Electrocardiography Not measured

A Drug plasma concentrations were evaluated in 3 additional groups: 28 males, 28 females in each group. Three animals/group/sex were bled at 4 time points (0.5, 4, 12 and 24 hours) after dosing on days 175 and 176. Body weights and observations for clinical signs of toxicity were also evaluated in these animals. Cmax and AUC increased with dose. Females had statistically significantly lower (p <0.05) plasma AUC values than did males at all dose levels.

B 6 animals died during the study. Based on the clinical signs, gross and/or histopathological findings, the deaths were considered unrelated to treatment. Two additional deaths occurred in the toxicokinetic groups. The cause of death was undetermined in these 2 males (120 mg/kg/day, 600 mg/kg/day) and the deaths were considered unrelated to treatment since no treatment-related deaths occurred at 3000 mg/kg/day in the toxicokinetic groups.

2.6.7.7.D. Repeat-Dose Toxicity

Report Title: A Six Month Oral Toxicity Study (With a Three Month Interim Sacrifice) in Mice Given 524W91



Daily Dose (mg/kg) 0 (Control) 120 600 3000 Number of Animals M: 25 F: 25 M: 25 F: 25 M: 25 F: 25 M: 25 F: 25 Hematology Very slight and reversible changes in total erythrocyte count (RBC), mean corpuscular volume (MCV), mean

corpuscular hemoglobin (MCH), and red cell distribution width (RDW) relative to control values were observed in male and female mice given 3000 mg/kg/day. These changes were present after approximately 3 months of dosing and were no greater in severity after 6 months of dosing.

Total erythrocyte count D t ns D t ns Mean corpuscular volume I t ns I t ns Mean corpuscular hemoglobin I t ns I t ns Red cell distribution width I t ns I t ns

Serum Chemistry No treatment related changes Urinalysis A very slight reversible increase in urine quantity was noted on dose day 179 in male and female mice given

3000 mg/kg/day. However, no treatment-related microscopic changes were observed in the kidney to correlate with this finding.

Organ Weights (%) There was a reversible increase in absolute and relative thyroid weights in female mice given 3000 mg/kg/day. Values were within control ranges and there were no histological changes to correlate with this finding.

Gross Pathology No treatment related changes Histopathology No treatment related changes Additional Examinations Electron microscopy – liver, heart, and skeletal muscle (from terminal sacrifice control and high-dose mice; first

3/sex/group) - No treatment related changes Postdose Evaluation: 3 weeks (5 mice/sex were randomly selected before the start of dosing for a post-dose recovery period. These mice

were bled for clinical pathology at the end of the recovery period). D= decrease I = increase p = permanent t = transitory ns = not significant

2.6.7.7.E. Repeat-Dose Toxicity Report Title: Three-Month Repeated Dose Oral Toxicity Study in the Mouse

Test Article: Rilpivirine


2.6.7.7.E. TMC278-NC119: Three-Month Repeated Dose Oral Toxicity Study in the Mouse

Species/strain: Mouse/SPF Swiss Age at first dose: 42 days Duration of dosing: 91 days Duration of postdose: /

Administration route/method: Oral/gavage Doses: 0 (vehicle) 20, 80 and 320 mg base eq./kg/day TMC278.HCl Test article batch: Vehicle/formulation: 0.5% w/v HPMC

Testing facility: J&J PRD Study no.: TMC278-NC119 GLP compliance: Yes Date of First Dose: - -20

No Observed Adverse Effect Level: 20 mg base eq./kg/day Toxicokinetics: Dose (mg base eq./kg/day) 0 (vehicle) 20 80 320 No. of animals M:12 F:12 M:15 F:15 M:15 F:15 M:15 F:15 Day 1 Cmax ( g/mL) - - 14 13 28 32 63 55 AUC0- ( g.h/mL) - - 71 59 236 250 1010 707 Day 31 Cmax ( g/mL) - - 14 18 38 37 63 84 AUC0-24 ( g.h/mL) - - 61b 74 263 313 860 1170 Day 87 Cmax ( g/mL) - - 18 19 34 42 61 90 AUC0-24 ( g.h/mL) - - 80 61 210 313 665 1360

Noteworthy Findings: Dose (mg base eq./kg/day) 0 (vehicle) 20 80 320 M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 Died or Sacrified Moribund 0 0 0 0 0 0 0 0 Body Weighta Week 13 / Day 91 35.3 29.1 - - - - 1.110** 1.110** Body Weight Gaina Week 13 / Day 91 6.6 6.2 - - - - 1.545*** 1.532** Food Consumptiona Total Food Consumption 473 489 - - 1.131** - 1.199*** 1.223** Clinical Observations Abdominal Distension 0 0 - - - - 5* 7** Waste of food - 0 - - - - - 3 a At the end of dosing period. For vehicle, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data

(not on the multiples of control/baseline); - = No noteworthy findings; * - p<0.05, ** - p<0.01, *** - p<0.001. b = AUC0-24h was more than 25% extrapoled; AUC0-8h = 61 g.h/mL; HPMC: hydroxypropyl methylcellulose.




a At the end of dosing period. For vehicle, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data (not on the multiples of control/baseline); - = No noteworthy findings; * - p<0.05, ** - p<0.01, *** - p<0.001

Report Title: TMC278-NC119: 3-Month Repeated Dose Oral Toxicity Study in the Mouse (continued)

Noteworthy Findings: Dose (mg base eq./kg/day) 0 (vehicle) 20 80 320 No. of animals M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 Hematologya White blood cells (10^3/ L) 7.6 - - - - - 0.526*** - Red blood cells (10^6/ L) 9.54 9.54 - - - - 0.917** 0.907** Hemoglobin (g/dL) 14.2 14.5 - - - - 0.923** 0.924** Hematocrit (%) 46.3 47.3 - - - - 0.940** 0.926*** Reticulocytes (10^3/ L) - 256.1 - - - - - 1.369* Thrombocytes (10^3/ L) - 1387 - - - - - 0.882* Neutrophils (10^3/ L) 1.28 - - - - - 0.695* - Lymphocytes (10^3/ L) 5.89 - - - - - 0.494*** - Eosinophils (10^3/ L) 0.21 - - - - - 0.524* - Serum Chemistrya Calcium (mg/dL) 9.0 9.1 - - - - 1.067* 1.077** Inorg. Phosphate (mg/dL) 7.2 - - - - - 1.181** - Total protein (g/dL) - 5.0 - - - 1.060* - 1.140*** Albumin (g/dL) 3.4 3.6 - - - 1.083*** 1.059* 1.111** Cholesterol (mg/dL) - 82 - - - 1.293* - 1.415* Triglycerides (mg/dL) - 158 - - - 0.620* - 0.386*** Urea nitrogen (mg/dL) - 24.1 - - - - - 1.249** Alk.phosphatase (U/L) 60 85 - - - - 2.250*** 2.976** Alanine aminotransferase (U/L) 88 63 - - - - 1.966 2.540***




Report Title: TMC278-NC119: 3-Month Repeated Dose Oral Toxicity Study in the Mouse (continued) Noteworthy Findings: Dose (mg base eq./kg/day) 0 (vehicle) 20 80 320 No. of animals M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 Organ Weightsa,b Liver (g) 1.81 1.37 - - 1.204* 1.372** 2.066** 2.664** Spleen (g) - 0.103 - - - - - 1.666 Gross Pathology Kidneys 10 10 10 10 10 10 10 10 - Irregular surface - 0 - - - - - 3 Liver 10 10 10 10 10 10 10 10 - Dark 0 0 - - - 4 10 10 - Swollen 0 0 - - - - 10 10 Spleen 10 10 10 10 10 10 10 10 - Swollen - 0 - - - - - 3 Histopathology Adrenal glands (n° examined) 10 10 10 10 10 10 10 10 Swollen/dense inner cortical cells 0 - 0 - 1 - 3 -

grade 1 0 0 1 3 Prominent X-zone - 5 - 4 - 1 - 0

grade 1 5 3 0 0 grade 2 0 1 1 0

Degeneration brown - 4 - 5 - 5 - 6 grade 1 3 5 4 1 grade 2 1 0 1 5

a At the end of dosing period. For vehicle, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data (not on the multiples of control/baseline); b = Both absolute and relative weights differed from controls in the direction indicated. Number indicates multiple of control for the absolute organ weights; - = No noteworthy findings; * - p<0.05, ** - p<0.01, *** - p<0.001




Report Title: TMC278-NC119: 3-Month Repeated Dose Oral Toxicity Study in the Mouse (continued) Noteworthy Findings: Dose (mg base eq./kg/day) 0 (vehicle) 20 80 320 No. of animals M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 Histopathology (cont’d) Bone marrow, femur(n° examined) 10 10 10 10 10 10 10 10 Increased M/E ratio 0 0 0 0 0 0 1 4

grade 1 0 0 0 0 0 0 1 2 grade 2 0 0 0 0 0 0 0 1 grade 3 0 0 0 0 0 0 0 1

Cellularity - 10 - 10 - 10 - 10 grade 2 1 1 0 0 grade 3 8 9 10 6 grade 4 1 0 0 3 grade 5 0 0 0 1

Bone marrow, sternum (n° examined) 10 10 10 10 10 10 10 10 Cellularity - 10 - 10 - 10 - 10

grade 3 9 9 9 8 grade 4 1 1 1 1 grade 5 1

Increased M/E ratio 0 0 0 0 0 0 5 4 grade 1 0 0 0 0 0 0 5 2 grade 2 0 0 0 0 0 0 0 1 grade 4 0 0 0 0 0 0 0 1

- = No noteworthy findings.




Report Title: TMC278-NC119: 3-Month Repeated Dose Oral Toxicity Study in the Mouse (continued) Noteworthy Findings: Dose (mg base eq./kg/day) 0 (vehicle) 20 80 320 No. of animals M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 Histopathology (cont’d) Kidneys (n° examined) 10 10 - 10 - 10 10 10 Nephropathy - 0 - 0 - 0 - 5

grade 1 2 grade 2 2 grade 3 1

Glomerulopathy - 0 - 1 - 0 - 5 grade 1 0 1 0 3 grade 2 0 0 0 2

Inflammation capsule/adjacent tissue - 0 - 1 - 0 - 3 grade 1 0 1 0 2 grade 2 0 0 0 1

Inflammation exudative - 0 - 0 - 0 - 1 grade 3 0 0 0 1

Inflammation granulocytic - 0 - 0 - 0 - 2 grade 1 0 0 0 2

Fibrosis - 0 - 0 - 0 - 4 grade 1 0 0 0 3 grade 2 0 0 0 1

Single cell death - 0 - 0 - 0 - 2 grade 1 0 0 0 2

Dilatation tubule - 0 - 0 - 0 - 3 grade 1 0 0 0 3

Infiltration mononuclear cell - 3 - 4 - 4 - 7 grade 1 3 3 4 3 grade 2 0 1 0 3 grade 3 0 0 0 1

Basophilia tubule(s) - 4 - 7 - 3 - 9 grade 1 4 6 3 4 grade 2 0 1 0 1 grade 3 0 0 0 3 grade 4 0 0 0 1





Report Title: TMC278-NC119: 3-Month Repeated Dose Oral Toxicity Study in the Mouse (continued) Noteworthy Findings:

Dose (mg base eq./kg/day) 0 (vehicle) 20 80 320 No. of animals M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 Histopathology (cont’d) Liver (n° examined) 10 10 10 10 10 10 10 10 Vacuolization hepatocellular 0 0 0 0 0 0 2 5

grade 1 0 0 0 0 0 0 2 3 grade 2 0 0 0 0 0 0 0 2

Pigmentation Kupffer cells 0 0 0 0 0 1 3 7 grade 1 0 0 0 0 0 1 3 4 grade 2 0 0 0 0 0 0 0 3

Extramedullary hematopoiesis - 0 - 0 - 0 - 2 grade 1 0 0 0 1 grade 2 0 0 0 1

Necrosis, single cell 3 0 3 2 2 1 9 7 grade 1 3 0 3 2 2 1 8 7 grade 2 0 0 0 0 0 0 1 0

Hypertrophy hepatocellular 4 0 2 1 8 7 10 10 grade 1 4 0 2 1 8 7 0 1 grade 2 0 0 0 0 0 0 6 6 grade 3 0 0 0 0 0 0 4 3

Kupffer cell proliferation - 0 - 0 - 0 - 3 grade 1 0 0 0 3

Ovaries (n° examined) 10 10 10 10 CL generations 10 10 10 9

grade 1 0 0 0 2 grade 2 3 4 3 2 grade 3 7 6 7 5

CL eosinophilic 10 10 10 7 grade 1 3 2 1 0 grade 2 2 2 1 3 grade 3 0 2 1 1 grade 4 1 1 2 1 grade 5 4 3 5 2





Report Title: TMC278-NC119: 3-Month Repeated Dose Oral Toxicity Study in the Mouse (continued)

Noteworthy Findings: Dose (mg base eq./kg/day) 0 (vehicle) 20 80 320 No. of animals M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 Histopathology (cont’d) CL basophilic 10 10 8 6

grade 1 9 10 7 6 grade 2 1 0 1 0

Spleen (n° examined) 10 10 10 10 10 10 10 10 Extramedullary hematopoiesis 1 4 2 3 3 6 7 9

grade 1 1 3 2 3 3 4 5 3 grade 2 0 1 0 0 0 2 2 5 grade 3 0 0 0 0 0 0 0 1

Thymus (n° examined) 10 10 10 10 Involution - 1 - 2 - 0 - 3

grade 1 1 2 0 2 grade 2 0 0 0 0 grade 3 0 0 0 1

Uterus (n° examined) 10 10 10 10 Infiltration granulocytic 9 10 9 6

grade 1 6 8 8 3 grade 2 3 2 1 3

Electronmicroscopy Liver (n° examined) 2 1 3 2 Increase of number of single membrane-bound organelles indicative of peroxisomes

0 0 2 2


2.6.7.7.F. Repeat-Dose Toxicity Report Title: A 13-Week Oral Gavage Toxicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Mouse

Test Article: Tenofovir disoproxil fumarate


2.6.7.7.F. M990203: A 13-Week Oral Gavage Toxicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Mouse

Species/Strain: Mouse, Swiss Crl: CD-1 (ICR) BR

Duration of dosing: 13 weeks Study No: Report No. M990203

Initial Age: 6 weeks (approx.) Duration of post dose: None Date of First Dose: 20 Method of administration: Oral gavage Vehicle/Formulation: Suspension vehicle GLP Compliance: Yes Special Features: None No observable Effect Level: Could not be determined Daily Dose (mg/kg) 0 control 100 300 1000/600C Number of Animals M: 15 F: 15 M: 15+32B F: 15 M: 15+32B F: 15 M: 15+32B F:15+32B Toxicokinetics (AUC)ss From Study M990203-PK

Day 91 (AUC 0- ) g.h/mL - - 14.6 - 35.8 - 68.1 55.2 Noteworthy Findings Died or Sacrificed Moribund D 0 1 0 0 0 1 18 6 Body Weight (%) A 38.52g 30.11g 0 0 0 0 0 0 Food Consumption (%) A 39.6g/animal 36.8g/animal 0 0 0 0 0 0 Water Consumption Not measured Clinical Observations There were no clinical signs considered treatment-related Ophthalmoscopy There were no effects of treatment Electrocardiography Not measured A At last measurement. For control groups, means are shown. For treated groups, biologically significant or treatment-related differences (%) are shown. B 32 animals included in toxicokinetic analysis. C Dose reduced to 600 mg/kg on Day 9. D Deaths due to problems administering a viscous suspension.

2.6.7.7.F. Repeat-Dose Toxicity Report Title: A 13-Week Oral Gavage Toxicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Mouse



Daily Dose (mg/kg) 0 control 100 300 1000/600C Number of Animals M: 15 F: 15 M: 15+32B F: 15 M: 15+32B F: 15 M: 15+32B F: 15+32B Hematology There were no effects of treatment Clinical Chemistry There were no clear effects of treatment Urinalysis Not measured Organ Weights (%) No effects of treatment Gross Pathology There were no gross pathological findings that could be attributed to treatment Histopathology Kidneys - Minimal Tubular

Karyomegaly 0 0 0 4/15 6/15 9/15 8/15 14/15

Duodenum - Epithelial Hypertrophy

0 0 0 0 3/15 13/15 6/15 14/15

A At last measurement. For control groups, means are shown. For treated groups, biologically significant or treatment-related differences (%) are shown. B 32 male animals included in toxicokinetic analysis. C Dose reduced to 600 mg/kg on Day 9. D Deaths due to problems administering a viscous suspension.

2.6.7.7.G. Repeat-Dose Toxicity Report Title: A 3-Month Oral Gavage Study for Bioassay Dose Selection in CD Rats (Emtricitabine)



2.6.7.7.G. TOX097: A 3-Month Oral Gavage Study for Bioassay Dose Selection in CD Rats (Emtricitabine)

Species/Strain: Rat / CD Duration of Dosing: 90 days Study Number: TOX097 Initial Age: Approximately 6 weeks old Duration of Postdose: None Date of First Dose: 20 Method of Administration: Oral Vehicle/Formulation: Vehicle control: 0.5% aqueous methylcellulose GLP Compliance: Yes

No Observed Adverse-Effect Level: A NOEL of 600 mg/kg/day was established.

Daily Dose (mg/kg) 0 (Control) 120 600 3000 Number of Animals M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10

Toxicokinetics: A AUC0-24 ( g.h/mL) Day 2 N/A N/A 61.6 55.6 296 260 1413 1357

Day 90 N/A N/A 65.6 69.3 329 362 1275 1646 Cmax ( g/mL) Day 2 N/A N/A 15.3 18.1 59.2 73.2 211 225

Day 90 N/A N/A 17.5 21.0 77.9 120 222 323 Noteworthy Findings

Died or Sacrified Moribund 0 0 0 0 0 0 0 0 Body Weight (%) There were no treatment-related effects Food Consumption (%) There were no treatment-related effects Water Consumption () Not measured Clinical Observations There were no treatment-related effects Ophthalmoscopy Not measured Electrocardiography Not measured Hematology Slightly decreased RBC counts and hemoglobin values were observed in both sexes at 3000 mg/kg/day, which reached

statistical significance for females only. Serum Chemistry No biologically meaningful effects on clinical chemistry parameters were observed Urinalysis No biologically meaningful effects on urinalysis parameters were observed

A Drug Plasma Concentrations: Emtricitabine was rapidly absorbed after oral dosing. Values for tmax (1-2 hours) tended to increase and dose normalized Cmax tended to decrease with increasing dose suggesting a slower oral absorption rate at higher doses. Dose-normalised AUC0-24 remained relatively constant with increasing dose, suggesting a linear relationship between systemic exposure and daily dose over the range of 120–3000 mg/kg/day. Female rats tended to absorb emtricitabine more rapidly than males (Cmax values on day 90 were approximately 20-50% higher in females), however, gender related differences in systemic exposure (AUC0-24) were not observed. Cmax and AUC0-24 were generally higher on day 90 compared to day 2, consistent with t1/2, especially in females.

2.6.7.7.G. Repeat-Dose Toxicity Report Title: A 3-Month Oral Gavage Study for Bioassay Dose Selection in CD Rats (Emtricitabine)




Organ Weights (%) Gross Pathology Necropsy Histopathology

Pituitary and thyroid/parathyroid gland weights were decreased at 3000 mg/kg/day, however, there were no associated histopathological changes

Postdose Evaluation: None

2.6.7.7.H. Repeat-Dose Toxicity Report Title: One-Month Repeated Dose Oral Toxicity Study in the Rat



2.6.7.7.H. TMC278-Exp5692: One-Month Repeated Dose Oral Toxicity Study in the Rat

Species/strain: Rat / Sprague Dawley Age at first dose: approximately 6 weeks Duration of dosing: 4 weeks Duration of postdose: 0 days

Administration route/method: Oral/gavage Doses: 0 (water), 0 (vehicle), 10, 40 and 160 mg/kg/day TMC278 base. Test article batch: Vehicle/formulation: PEG400 + CA

Testing facility: Study no.: TMC278-EXP5692 GLP compliance: Yes Date of first dose: 20

No Observed Adverse Effect Level: 10 mg/kg/day Toxicokinetics: Dose (mg/kg/day) 0 (water) 0 (vehicle) 10 40 160 No. of animals M:0 F:0 M:0 F:0 M:4 F:4 M:4 F:4 M:4 F:4 Day 1 Cmax ( g/mL) - - - - 0.94 2.0 2.0 3.2 6.5 9.6 AUC0- ( g.h/mL) - - - - 7.5 14 23 30 52 77 Day 29 Cmax ( g/mL) - - - - 0.88 1.6 2.6 5.8 6.7 8.8 AUC0-24h ( g.h/mL) - - - - 7.2 14 27 42 51 89 Noteworthy Findings Dose (mg/kg/day) 0 (water) 0 (vehicle) 10 40 160 No. of animals M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 Died or Sacrified Moribund 0 0 0 0 0 0 0 0 0 0 Body Weight - - - - - - - - - - Body Weight Gain - - - - - - - - - - Food Consumption - - - - - - - - - - Clinical Observations - - - - - - - - - - Ophthalmoscopy - - - - - - - - - - - = No noteworthy findings; PEG400: polyethylene glycol; CA: citric acid.

2.6.7.7.H. Repeat-Dose Toxicity Report Title: One-Month Repeated Dose Oral Toxicity Study in the Rat



Report Title: TMC278-TOX5692: One-Month Repeated Dose Oral Toxicity Study in the Rat (continued) Noteworthy Findings

Dose (mg/kg/day) 0 (water) 0 (vehicle) 10 40 160 No. of animals M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 M:10 F:10 Hematology - - - - - - - - - - Serum analysisa Glucose (g/dL) - 0.96 - 0.88 - - - - - 0.898*** Urinalysis - - - - - - - - - - Organ weights (absolute)a Liver (g) 12.08 7.16 10.87 7.30 - - 1.149 1.105* 1.233 1.119* Thyroid (g) 0.021 0.020 0.017 0.016 - - 1.249 1.236 1.235 1.224 Pituitary gland (g) 0.011 0.014 0.010 0.013 - - 1.074 - 1.162 1.136 Histopathologya Thyroid Follicular hypertrophy 0 0 0 0 0 0 2 0 6 4 No. of animals M:8 F:8 M:8 F:8 M:8 F:8 M:8 F:8 M:8 F:8 PFC assay - - - - - - - - - - a For vehicle, group means are shown. For treated groups, multiples of vehicles are shown. Statistical significance is based on actual data (not on the multiples of

control/baseline). - = No noteworthy findings; * - p<0.05, *** - p<0.001; PFC: plaque forming cell.

2.6.7.7.I. Repeat-Dose Toxicity Report Title: Six-Month Repeated Dose Oral Toxicity Study with 1-Month Recovery in the Rat



2.6.7.7.I. TMC278-NC101: Six-Month Repeated Dose Oral Toxicity Study with 1-Month Recovery in the Rat

Species/strain: Rat / Sprague Dawley Age at first dose: 42 days Duration of dosing: 186 days Duration of postdose: 158 days

Administration route/method: Oral/gavage Doses: 0 (vehicle), 40, 120 and 400 mg/kg TMC278 base Test article batch: Vehicle/formulation: PEG400 + CA

Testing facility: J&J PRD. Study no.: TMC278-NC101 GLP compliance: Yes Date of first dose: 20

No Observed Adverse Effect Level: / Toxicokinetics: Dose (mg/kg/day) 0 (vehicle) 40 120 400 No. of animals M:3 F:3 M:6 F:6 M:6 F:6 M:6 F:6 Day 1 Cmax ( g/mL) - - 2.9 6.5 6.4 8.5 9.1 17 AUC0- ( g.h/mL) - - 19 32 53 83 92 160 Day 83 Cmax ( g/mL) - - 3.4 8.2 3.4 11 3.9 15 AUC0-24h ( g.h/mL) - - 19 41 41 100 57 184 Day 175 Cmax ( g/mL) - - 1.7 6.6 3.0 8.8 6.2 16 AUC0-24h ( g.h/mL) - - 12 50 35 116 73 244 Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 40 120 400 No. of animals M:30 F:30 M:20 F:20 M:20 F:20 M:30 F:30 Mortality 0 0 0 0 0 0 0 0 Clinical observations Salivation 15 5 15 14*** 14 13*** 25* 17** Wet urogenital region 7 2 - - - - 13 11* / = not determined; - = No noteworthy findings; PEG400: polyethylene glycol; CA: citric acid; * - p<0.05, ** - p<0.01, *** - p<0.001; M: male; F: female.




Report Title: TMC278-NC101: Six-Month Repeated Dose Oral Toxicity Study with 1-Month Recovery in the Rat (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 40 120 400 No. of animals M:30 F:30 M:20 F:20 M:20 F:20 M:30 F:30 Body weight - - - - - - - - Food consumption - - - - - - - - Ophthalmoscopy - - - - - - - - Hematologya Interim analysis (3 months) Act. Part. Thromb. Time (sec) 25.62 - 1.116*** - 1.230*** - 1.190*** - Prothrombine Time (sec) 20.52 - 1.125* - 1.274*** - 1.133* - End of dosing (6 months) Act. Part. Thromb. Time (sec) 20.85 - 1.148** - 1.249*** - 1.164*** - Prothrombine Time (sec) 18.82 - 1.134** - 1.272*** - 1.159** - Red blood cells (10^6/ L) 9.20 - - - - - 0.948** - Hemoglobin (g/dL) 16.4 - - - - - 0.951*** - Hematocrit (%) 48.6 - - - - - 0.955** - Mean cell volume (fL) - 55.1 - - - 0.976* - 0.978* Mean cell hemoglobin (pg) - 18.7 - - - 0.973* - 0.973* Eosinophils (10^3/ L) - 0.15 - 0.733** - 0.733** - 0.667*** End of recovery M:10 F:10 M:10 F:10 Act. Part. Thromb. Time (sec) 21.61 - - - - - 1.140* - Prothrombine Time (sec) 20.46 - - - - - 1.123* - a For controls, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data (not on the multiples of

control/baseline); - = No noteworthy findings; * - p<0.05, ** - p<0.01, *** - p<0.001; M: male; F: female.




Report Title: TMC278-NC101: Six-Month Repeated Dose Oral Toxicity Study with 1-Month Recovery in the Rat (continued) Noteworthy Findings:

Dose (mg/kg/day) 0 (vehicle) 40 120 400 No. of animals M:30 F:30 M:20 F:20 M:20 F:20 M:30 F:30

Serum Chemistrya Interim analysis (3months) Inorg. Phosphorus (mg/dl) 7.6 6.4 - 1.063* 1.026* 1.063* 1.105*** 1.078*** Total protein (g/dl) - 7.0 - - - - - 1.043** Albumin (g/dl) 4.5 4.9 - - 1.089*** 1.041** 1.089*** 1.082*** Triglycerides (mg/dl) 76 53 0.724** 0.849* 0.855 0.811* 0.632*** 0.755*** Total bilirubin (mg/dl) 0.12 0.13 0.833*** 0.769*** 0.750*** 0.692*** 0.667*** 0.769*** Alkaline phosphatase (U/l) 79 - - - 1.291*** - 1.532*** - End of dosing (6 months) Calcium (mg/dl) 11.7 - - - - - 0.957** - Total protein (g/dl) 7.4 7.7 - - - - - 1.039** Albumin (g/dl) 4.5 5.2 - - 1.067*** - 1.067*** 1.077*** Glucose (mg/dl) 88 - 0.886** 0.886** Triglycerides (mg/dl) 91 83 - - - - 0.681** 0.590*** Urea nitrogen (mg/dl) 18.1 - - - 1.099* - 1.099* - Creatinine (mg/dl) 0.26 - 1.115* - 1.115** - 1.115* - Total bilirubin (mg/dl) 0.13 0.15 0.769** 0.667*** 0.692*** 0.600*** 0.615*** 0.667*** Alkaline phosphatase (U/l) 69 - - - 1.319** - 1.696*** - Urinalysisa End of recovery M:10 F:10 M:10 F:10 Specific gravity 1.032 - 1.005* - Ketones (grade) 0.67 - 2.493* - a For controls, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data (not on the multiples of

control/baseline); - = No noteworthy findings * - p<0.05, ** - p<0.01, *** - p<0.001




Report Title: TMC278-NC101: Six-Month Repeated Dose Oral Toxicity Study with 1-Month Recovery in the Rat (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 40 120 400 No. of animals M:30 F:30 M:20 F:20 M:20 F:20 M:30 F:30 Endocrinologya End of dosing TSH (ng/ml) 4.7 3.9 1.447** 1.769*** 1.766* 2.410*** 1.234* 2.487*** T4 (nmol/l) 44 32 0.636** 0.750* 0.432*** 0.563*** 0.545*** 0.563*** T3 (nmol/l) 0.82 1.2 0.707* - - - 1.341* - Corticosterone (nmol/l) 1065 1291 - - 0.922 0.775* 0.823 0.914 Progesterone (nmol/l) 11 39 - - 1.727 - 1.909 1.385** ACTH (pg/mL) - - - - - - - - End of recovery M:10 F:10 M:10 F:10 T4 (nmol/l) - 32 - 1.375** Organ Weightsa,b End of dosing Liver (mg) 13036 7883 - - 1.207** 1.110** 1.246*** 1.239*** Thyroids (mg) 24 19 1.208** 1.158* 1.375*** 1.368*** 1.500*** 1.526*** End of recovery M:10 F:10 M:10 F:10 Thyroids (mg) - 18 - 1.389** Gross Pathology End of dosing Liver (n° examined) 20 20 20 20 20 20 20 20 - dark 0 0 - - - - 2 3 - yellow focus 0 - - 3 - more pronounced lobulation 0 0 - - 2 - 7* 5 - swollen 1 - - - - - 5 Thyroid glands (n° examined) 20 20 20 20 20 20 20 20 - swollen 0 0 - - 2 3 6* 9** a For controls, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data (not on the multiples of control/baseline); b = Both absolute and relative weights differed from controls in the direction indicated. Number indicates multiple of control for the absolute organ weights; - = No noteworthy findings; * - p<0.05, ** - p<0.01, *** - p<0.001




Report Title: TMC278-NC101: Six-Month Repeated Dose Oral Toxicity Study with 1-Month Recovery in the Rat (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 40 120 400 No. of animals M:30 F:30 M:20 F:20 M:20 F:20 M:30 F:30 Histopathology Liver (n° examined) 19 19 20 20 20 20 19 20 - hypertrophy 3 - 3 7** 8 16*** 16***

Grade 1 - 2 - 3 7 8 6 10 Grade 2 - 1 - 0 0 0 10 6

Lymph nodes, mesenteric (n° examined) 19 19 19 19 19 19 19 19 - swollen-vacuolated small macrophage-aggregates

- - - - - - 5* 4

Grade 1 - - - - - - 4 4 Grade 3 - - - - - - 1 0

Pituitary gland (n° examined) 18 - 18 - 19 - 19 - - large vacuolated cells/swollen cells (pars distalis)

2 - 11** - 12** - 9* -

Grade 1 2 - 10 - 10 - 9 - Grade 2 - 1 - 2 - - -

Thyroid glands (n° examined) 17 19 16 18 19 19 18 19 - high follicular epithelium 2 - 4 12*** 15*** 19*** 18*** 19***

Grade 1 2 - 4 12 14 16 5 2 Grade 2 - - - 1 3 13 17

- small follicles 4 3 12** 17*** 18*** 18*** 11* 17*** Grade 1 4 3 6 12 6 5 7 2 Grade 2 0 - 6 5 12 13 4 10 Grade 3 0 0 0 0 0 0 0 5

- = No noteworthy findings; * - p<0.05, ** - p<0.01, *** - p<0.001




Report Title: TMC278-NC101: Six-Month Repeated Dose Oral Toxicity Study with 1-Month Recovery in the Rat (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 40 120 400 No. of animals M:10 F:10 M:10 F:10 End of recovery - - - - Lymph nodes, mesenteric (n° examined) 9 9 6 10 - swollen-vacuolated small macrophage-aggregates

0 0 2 3

Grade 1 2 2 Grade 2 0 1

Thyroid glands (n° examined) 9 9 6 10 - high follicular epithelium 0 0 2 0

Grade 1 0 2 - small follicles 4 1 6* 5

Grade 1 3 0 4 3 Grade 2 1 1 2 2

- = No noteworthy findings; * - p<0.05.

2.6.7.7.J. Repeat-Dose Toxicity Report Title: A 14-day oral gavage toxicity study of bis-POC PMPA fumarate (GS-4331-05; PMPA prodrug) in the albino rat



2.6.7.7.J. 98-TOX-4331-004: A 14-Day Oral Gavage Toxicity Study of bis-POC PMPA Fumarate (GS-4331-05, PMPA Prodrug) in the Albino Rat

Species/Strain: Rat, Sprague Dawley CD (Crl: CD-1 (SD) BR

Duration of dosing: 14 days Study No: 98-TOX-4331-004

Initial Age: 9 weeks (approx) Duration of post dose: 1 day Date of First Dose: 19 Method of administration: Oral gavage Vehicle/Formulation: Suspension vehicle. GLP Compliance: Yes Special Features: None No observable Effect Level: 500 mg/kg Daily Dose (mg/kg) 0 Control 500 750 1000 1250 Number of Animals M: 5 M: 5 M: 5 M: 5 M: 5 Toxicokinetics (AUC)ss From Study 98-TOX-4331-004-PK

AUC not calculated. All animals that received TDF demonstrated exposure to tenofovir based upon measurable levels of tenofovir in the plasma on days 1 and 14. Plasma tenofovir concentrations on day 1 were not dose proportional with dose, suggesting saturation of drug absorption.

Noteworthy Findings Died or Sacrificed Moribund 0 0 1 2 5 Body Weight (%) 394.4g 0 -17 -23 -39 Food Consumption - Significantly lower

days 1-5 Significantly lower

days 1-11 [excluding day 7-8]

Significantly lower days 2-11

[excluding day 8-9]

Significantly lower days 1-9

Water Consumption Not measured Clinical Observations Salivation - 5/5 5/5 5/5 5/5 Soft Feces - - 2/5 1/5 2/5 Liquid Feces - - - 1/5 1/5 Black Feces - - 1/5 - 1/5 Generalized Ungroomed&/or Erect

Fur - - 4/5 5/5 3/5

Wet Fur/Genital Region - - - 1/5 - Dehydration - - 4/5 5/5 4/5 Eyes Partly Closed - - 1/5 1/5 2/5

2.6.7.7.J. Repeat-Dose Toxicity Report Title: A 14-day Oral Gavage Toxicity Study of bis-POC PMPA Fumarate (GS-4331-05; PMPA Prodrug) in the Albino Rat



Daily Dose (mg/kg) 0 Control 500 750 1000 1250 Number of Animals M: 5 M: 5 M: 5 M: 5 M: 5 Clinical Observation (cont) Decreased Activity - - 1/5 1/5 3/5 Cold to Touch - - - 1/5 2/5 Weak - - - 2/5 1/5 Respiratory Rate Irregular - - - - 1/5 Reduced Appetite - - - 1/5 - Lying on Side - - - 1/5 1/5 Ophthalmoscopy Not measured Electrocardiography Not measured Hematology White Cell Counts - - I I - Segmented Neutrophil Counts - - I I - % Lymphocyte Counts - - D D - Clinical Chemistry AST - - I I - ALT - - I I - Urinalysis Not measured Organ Weights Adrenal - - I I - Thymus - - D D -

2.6.7.7.J. Repeat-Dose Toxicity Report Title: A 14-day Oral Gavage Toxicity Study of bis-POC PMPA Fumarate (GS-4331-05; PMPA Prodrug) in the Albino Rat



Daily Dose (mg/kg) 0 Control 500 750 1000 1250 Number of Animals M: 5 M: 5 M: 5 M: 5 M: 5 Gross Pathology Dilation of the Ileum - - - - 1/5 Dilation of the Jejunum - - - - 2/5 Stomach Dilatation, Areas of

Muscosal Thickening Area Raised or Depressed Areas, Areas of Discolouration

- - - 3/5 5/5

Thickening of Gastric Mucosa - - - 2/5 - Thickening of Duodenal Wall - - 1/5 1/5 - Small thymus - - 1/5 2/5 2/5 Small Spleen - - - 1/5 4/5 Histopathology Gastritis - - - 1/5 - Stomach Erosion Glandular - - - - 4/5 Stomach Ulcer, Glandular - - - - 1/5 Stomach Ulcer, Squamous - - - 2/5 4/5 Stomach Hyperplasia, Squamous - - 1/5 3/5 3/5 Duodenitis - - 1/5 1/5 2/5 Duodenal Glandular Dilation - - - - 3/5 Duodenal Erosion - - 1/5 1/5 - Duodenal, Hyperplasia, Muscosal - - 4/5 3/5 3/5 Ileitis - - - - 2/5 Jejunum Congestion, Muscosal - - - - 1/5 Jejunitis - - - 1/5 3/5 Thymus Atrophy, Lymphoid - - 2/5 3/5 4/5 Spleen Atrophy, Lymphoid - - 1/5 1/5 5/5

2.6.7.7.K. Repeat-Dose Toxicity

Report Title: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 in the Albino Rat


NC not calculated due to insufficient data. A= At last measurement. For controls group means are shown. For treated groups, biologically significant or treatment-related differences (%) from controls are shown. B =Accidental death gavage error. C= As change was not accompanied by histopathological changes it is of uncertain biological significance. $= value within historically acceptable range for animals of this strain and age. CONFIDENTIAL Page 112 17AUG2010

2.6.7.7.K. 96-TOX-4331-003: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 in the Albino Rat

Species/Strain: Rat Sprague Dawley CD (Crl: CD (SD) BR)

Duration of Dosing: 28 days Study No: 96-TOX-4331-003

Initial Age: 7 weeks (approx.) Duration of Post Dose: 1 day Date of First Dose: 19

Method of Administration: Oral gavage

Vehicle/Formulation: Carboxymethylcellulose, benzyl alcohol, polysorbate 20 and NaCl

GLP Compliance: Yes

Special Features: None No Observable Effect Level: 100 mg/kg/day 500 mg/kg/day Daily Dose (mg/kg) 0 control 20 100 500 Number of Animals M: 10 F: 10 M: 18 F: 10 M: 18 F: 10 M: 18 F: 18 Toxicokinetics (AUC)ss From Study 96-TOX-4331-003-PK

Day 28 (AUC 0- ) μg.hr/mL - - NC NC NC NC 17.0 13.7 Noteworthy Findings Died or Sacrificed Moribund 0 0 0 0 0 0 0 1B Body Weight (%) A 366.9g 252.7g 0 0 0 0 0 0 Food Consumption (%) A 196.3 g

/animal 161.6 g /animal

0 0 0 0 0 0

Water Consumption Not measured Clinical observations There were no clinical signs considered treatment-related Ophthalmoscopy Not measured Electrocardiography Not measured

2.6.7.7.K. Repeat-Dose Toxicity

Report Title: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 in the Albino Rat


A At last measurement. C= As change was not accompanied by histopathological changes it is of uncertain biological significance. For controls group means are shown. For treated groups, biologically significant or treatment-related differences (%) from controls are shown. $= value within historically acceptable range for animals of this strain and age.

* P<0.01, ** p< 0.05, I= Increase, D= decrease, + = mild CONFIDENTIAL Page 113 17AUG2010

Daily Dose (mg/kg) 0 control 20 100 500 Number of Animals M: 10 F: 10 M: 18 F: 10 M: 18 F: 10 M: 18 F: 18 Hematology Red Cell Distribution Width - - - - - - +I*$ - Hemoglobin - - - - - - +D*$ +D*$ Hematocrit - - - - - - +D**$ - Mean Corpuscular Volume - - - - - - +D*$ - Mean Corpuscular Hemoglobin - - - - - - +D*$ - Mean Corpuscular Hemoglobin

Concentration - - - - - - +D*$ -

Clinical Chemistry ALT - - - - - - +I* +I* Total Protein - - - - - - - +D* Albumin - - - - - - - +D* Sodium - - - - - - - +D** Urinalysis Not measured Organ Weights (%)A Absolute/Relative Kidney Weight 2.605g 1.809g 0 0 0 0 -12*C 0 Gross Pathology There were no gross pathological findings that could be attributed to treatment. Histopathology There were no histopathological findings that could be attributed to treatment.

2.6.7.7.L. Repeat-Dose Toxicity

Report Title: A 13- and 42-Week Oral Gavage Toxicity Study (with a 13-Week Recovery Period) of Bis-POC PMPA (GS-4331-05) in the Albino Rat


A At last measurement during treatment day 294. For controls group means are shown. For treated groups, biologically significant or treatment-related differences (%) from controls are shown. B= Mixed male and female value. C=None of these deaths were considered treatment related. E= Mean data for days 162–169, a decrease in food consumption was seen in males in 1000 mg/kg group up to day 169 compared to controls. F= No changes in organ weights were considered toxicologically significant

* P 0.01, ** p 0.05, + = mild, ++ moderate, +++ severe, D=decrease, I=increase, t=transitory. CONFIDENTIAL Page 114 17AUG2010

2.6.7.7.L. 97-TOX-4331-002: A 13- and 42-Week Oral Gavage Toxicity Study (with a 13-Week Recovery Period) of Bis-POC PMPA (GS-4331-05) in the Albino Rat

Species/Strain: Rat, Sprague Dawley CD (Crl: CD (SD) BR)

Duration of Dosing: 13 or 42 weeks Study No: 97-TOX-4331-002

Initial Age: 6 weeks (approx.) Duration of Post Dose: 13 weeks Date of first dose: 19 Method of Administration: Oral gavage Vehicle/Formulation: Carboxymethylcellulose sodium, benzyl alcohol, GLP Compliance: Yes Special Features: None polysorbate 20 & sterile water. No Observable Effect Level: Could not be determined Daily Dose (mg/kg) 0 Control 30 100 300 1000 Number of Animals (10 13 wk cohort, 10 42 wk cohort & 5 recovery animals per sex per group, plus 8 TK animals per sex in TDF treated groups) M: 25 F: 25 M: 33 F: 33 M: 33 F: 33 M: 33 F: 33 M: 33 F: 33 Toxicokinetics (AUC)ss From Study 97-TOX-4331-002-PK

Week 13 (AUC0- ) μg.h/mL - - 2.65B 2.65B 7.00B 7.00B 18.4B 18.4B 34.1B 34.1B Week 26 (AUC0- ) μg.h/mL - - 3.93B 3.93B 6.89B 6.89B 13.8B 13.8B 42.8B 42.8B Week 42 (AUC0- ) μg.h/mL - - 3.78B 3.78B 8.34B 8.34B 17.6B 17.6B 64.9B 64.9B Noteworthy Findings Died or Sacrificed MoribundC 0 2 1 1 0 2 1 0 4 4 Body Weight (%) A, 670.7g 354.6g 0 0 0 0 0 0 -26* 0 Food Consumption (%) E 210.3g 138.7g 0 0 0 0 0 0 -17* 0 Water Consumption Not measured Clinical Observations Salivation - - +t +t + + +/++ +/++ ++/+++ ++/+++ Ophthalmoscopy There were no treatment related ocular changes throughout the study Electrocardiography Not measured


Report Title: A 13- and 42-Week Oral Gavage Toxicity Study (with 13-Week Recovery Period) of Bis-POC PMPA (GS-4331-05) in the Albino Rat




Daily Dose (mg/kg) 0 Control 30 100 300 1000 M: 25 F: 25 M: 33 F: 33 M: 33 F: 33 M: 33 F: 33 M: 33 F: 33 Hematology RBC - - - - I - I - I - WBC and RBC Distribution Width - - - - - - - - I - Reticulocyte - - - - - - - - - I Segmented Neutrophil - - - - - - - - - I Hematocrit - - - - - - - - - D Hemoglobin - - - - - - - - - D MCV - - - - - - - - - D MCH - - - - - - - - - D MCHC - - - - - - - - - D MPV - - - - - - - - - D Platelet Counts - - - - - - - I - I Clinical chemistry Cholesterol - - - - D - D - D - ALT - - - - - - I - I - AST - - - - - - I - I - Phosphorus - - - - - - I - I - A/G Ratio - - - - - - I - I - Total Protein - - - - - - D - - D Globulins - - - - - - D - - D Triglycerides - - - - - - D - - D Bicarbonate - - - - - - - D - D Creatinine - - - - - - - - - I Urinalysis There were no clear treatment related effects on urinalysis parameters






Daily Dose (mg/kg) 0 Control 30 100 300 1000 M: 25 F: 25 M: 33 F: 33 M: 33 F: 33 M: 33 F: 33 M: 33 F: 33 Bone Decreases in Cortical/Subcortical Area,

Bone Mineral Content, and Density (correlated with effects on circumference)

- - - - - - D - D D

Osteocalcin - - - - - - I - I I Urinary Deoxypyridinoline - - - - I I I I I I Urinary Calcium - - - - - - - - I I Urinary Phosphorus - - - - - - - I I PTH - - - - - - - - - I Organ weights Adrenal Weights - - - - - - - - I I Gross & Histopathological Findings Renal Tubular Karyomegaly - - + + + + + + + + Renal Tubular Pigment Accumulation - - + + + + + + +/++ +/++ Epithelial cell Hypertrophy in the

Duodenum - - - - + + + + + +

Epithelial Cell Hypertrophy in the Jejunum

- - - - - - - - + +

Gastritis (ulcerative) - - - - - - + + + + Typhlitis - - - - - - + + +/++ +/++ Mucosal Hyperplasia in the Duodenum - - - - - - + + +/++ +/++ Jejunitis (ulcerative and/or

hyperkaratosis) - - - - - - - - + +

Colitis - - - - - - + + + + Duodenitis - - - - - - - - ++ ++ Ileitis - - - - - - - - + +






Daily Dose (mg/kg) 0 Control 30 100 300 1000 M: 25 F: 25 M: 33 F: 33 M: 33 F: 33 M: 33 F: 33 M: 33 F: 33

Gross & Histopathological Findings (Continued)

Atrophy of the Cecum - - - - - - - - +/++ +/++ Atrophy of the Colon - - - - - - - - + + Villous Atrophy in the Ileum - - - - - - - - + +

Post Dose Evaluation 13 week recovery (n=5 males/females per group) Renal Tubular Karyomegaly - - - - - - + + + +

Renal Tubular Pigment Accumulation

- - - - + + + + + +

Typhlitis - - - - - - + + - - PTH - - - - - - - - - I Osteocalcin - - - - - - - - I -

2.6.7.7.M. Repeat-Dose Toxicity Report Title: A 14-Day Oral Gavage Toxicity Study Comparing Non-Degraded and Degraded TDF/FTC in Sprague-Dawley Rats

Test Article: Emtricitabine/Tenofovir DF


2.6.7.7.M. TX-164-2001: A 14-Day Oral Gavage Toxicity Study Comparing Non-Degraded and Degraded TDF/FTC in Sprague-Dawley Rats

Species/Strain: Rat/Sprague-Dawley CD® (Crl: CD®(SD)BR)

Duration of Dosing: 14 Days Study No TX-164-2001

Initial Age: 7 to 8 Weeks Duration of Postdose: N/A Date of First Dose: , 20 Method of Administration: Oral Gavage Vehicle/Formulation: Suspension Vehicle

(carboxymethylcellulose, 0.5% (w/w), benzyl alcohol, 0.9% (w/w), polysorbate 20, 0.5% (w/w) and sterile water for injection, USP).

GLP Compliance: Yes Special Features: No females on study No Adverse Effect Dosage Level: 100/67 mg/kg/day Dose (mg/kg/day) TDF/FTC 0

(control) 30/20

non-degraded 100/67

non-degraded 300/200

non-degraded 30/20 degraded 100/67

degraded 300/200

degraded Number of Animals /Group M: 10 M: 10 M: 10 M: 10 M: 10 M: 10 M: 10 Noteworthy Findings Died or Euthanized Moribund 0 0 0 0 0 0 0 Body Weight A 325 g - - - - - - Food Consumption A 25 g/day

/animal - - - - - -

Clinical Observations Number of Animals Observed

Salivation (post-dose) 0/10 0/10 10/10 10/10 2/10 10/10 10/10 Hematology - - - - - - - Serum Chemistry - - - - - - - Urinalysis - - - - - - - Absolute Organ Weight

Adrenal glands (g) 0.047 0.058 (+23%)** 0.057 (+21%)* 0.057 (+21%)** 0.054 0.056 (+19%)*

0.056 (+19%)*

A At last measurement % differences not calculated for values considered to be comparable to controls or not scientifically relevant. - no significant treatment-related findings * p 0.05; ** p 0.01

2.6.7.7.M. Repeat-Dose Toxicity Report Title: A 14-Day Oral Gavage Toxicity Study Comparing Non-Degraded and Degraded TDF/FTC in Sprague-Dawley Rats

Test Article: Emtricitabine/Tenofovir DF


Dose (mg/kg/day) TDF/FTC 0 (control)

30/20 non-degraded

100/67 non-degraded

300/200 non-degraded

30/20 degraded 100/67 degraded

300/200 degraded

Number of Animals /Group M: 10 M: 10 M: 10 M: 10 M: 10 M: 10 M: 10 Relative Organ Weight

Adrenal glands (% body weight)

0.016 0.019 (+19%)** 0.018 (+13%)* 0.019 (+19%)** 0.018 0.018 0.019 (+19%)*

Gross Pathology - - - - - - - Histopathology

Anterior Duodenal Mucosa Overlying Brunner’s Glands

- Hyperplasia 0/10 0/10 0/10 7/10 0/10 0/10 2/10

- no significant treatment-related findings. * p 0.05; ** p 0.01

2.6.7.7.N. Repeat-Dose Toxicity Report Title: One-Month Repeated Dose Oral Toxicity Study in the Dog with 1-Month Recovery



2.6.7.7.N. TMC278-Exp5650: One-Month Repeated Dose Oral Toxicity Study in the Dog with 1-Month Recovery

Species/strain: Dog/Beagle Age at first dose: approximately 6 to 8 months Duration of dosing: 1-month Duration of postdose: 1-month

Administration route/method: Oral/Gavage Doses: 0 (water), 0 (vehicle), 5, 10, 40 mg/kg/day TMC278 base Test article batch: Vehicle/formulation: PEG400 + CA

Testing facility: J & J PRD Study No.: TMC278-Exp5650 GLP compliance: Yes Date of first dose: 20

No Observed Adverse Effect Level: / Toxicokinetics

Doses (mg/kg/day) 0 (control) 5 10 40 No. of Animals M:3 F:3 M:3 F:3 M:3 F:3 M:3 F:3 Day 1 Cmax ( g/mL) - - 0.94 1.3 1.3 1.3 2.8 2.4 AUC0-24h ( g.h/mL) - - 13 15 22 14 51 40 Day 28 Cmax ( g/mL) - - 1.5 2.0 5.6 2.5 12 9.5 AUC0-24h ( g.h/mL) - - 27 37 103 47 204 160

Noteworthy Findings

0 (control) 0 (vehicle) 5 10 40 Doses (mg/kg/day) No. of Animals M:3 F:3 M:3 F:3 M:3 F:3 M:3 F:3 M:3 F:3 Mortality 0 0 0 0 0 0 0 0 0 0 Clinical observations - - - - - - - - - - ECG - - - - - - - - - - Eye examination - - - - - - - - - - Body Weight (g) 12.0 10.5 12.6 10.9 - - - 0.917 0.976 0.917 Food Consumption (kg) 1.96 0.32 2.31 0.35 - 0.743 0.848 0.771. 0.710 0.857 Hematologyb Hematocrit (%) 50.6 47.4 - - 0.973 Hemoglobin (g/dL) 16.5 15.4 - - 0.968 Red blood cells (10^3/ l) 7.02 6.49 - - 0.980 White blood cells (10^3/ l) 8.77 9.18 - - 1.121




0 (control) 0 (vehicle) 5 10 40 Doses (mg/kg/day) No. of Animals M:3 F:3 M:3 F:3 M:3 F:3 M:3 F:3 M:3 F:3 Serum analysisb Albumin (g/dL) 3.8 3.7 - 0.973 0.973 Total protein (g/dL) 6.2 6.0 - 0.983 0.983 Cholesterol (mg/dL) 135 128 - - 1.320** Triglycerides (mg/dL) 32 28 - 0.857 0..786 Total bilirubin (mg/dL) 0.13 0.11 - - 2.363*** Alkaline phosphatase (U/L) 111 138 - - 2.210** Alanine aminotransferase (U/L) 47 44 - - 2.932* a For controls, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data (not on the multiples of control/baseline); b = For hematology and serum analysis, males and female values are combined; - = No noteworthy findings; / = not determined; * - p<0.05, ** - p<0.01, *** - p<0.001; F = female, M = male; / = not determined; PEG400: polyethylene glycol; CA: citric acid




Report Title: TMC278-Exp5650: One-Month Repeated Dose Oral Toxicity Study in the Dog with 1-Month Recovery (continued) Noteworthy Findings Doses (mg/kg/day) 0 (control) 0 (vehicle) 5 10 40 No. of animals M:3 F:3 M:3 F:3 M:3 F:3 M:3 F:3 M:3 F:3 Urinalysis - - - - - - - - Endocrinology ACTH (pg/mL.h) 965 737 521 785 - - 1.710 1.237 3.77 2.964 Cortisol (nmol/L.h) 1386 1521 674 1256 - - - 0.557 - 0.581 Progesterone (nmol/L) 0.81 0.86 0.76 0.84 - - 7.500 17.86 28.95* 11.43* Organ weights Ovaries 0.826 0.771 1.326 2.597 2.384* Gross pathology Swollen cervix 0 1 0 2 2 Swollen ovaries 0 1 0 2 2 Swollen uterus 0 1 0 2 2 Swollen vagina 0 1 0 2 2 Swollen mammary gland 0 0 0 0 0 0 0 1 0 0 Histopathology Adrenal cortex Reduced vacuolation diffuse (z.f.) - - - - - - P P P P Largely vacuolated cells, multifocal (z.f.) - - - - - - P - P - Mixed leucocytic infiltrates (z.f.) - - - - - - - - P - Swollen cells (z.f./z.r.) - - - - - - - P P P Female genital tract Prominent activation - - - - - - - P - P Liver (sub)chronic centrilobular perivascular inflammation

- - - - - - P P P P

Mammary gland Prominent activation - - - - - - P P P P Recovery - - - - - - - - liver liver F = female; M = male; - = No noteworthy findings; P = present; ACTH = Adreno CorticoTropic Hormone; z.f. = zona fasciculate; z.r. = zona reticularis; * - p<0.05, ** - p<0.01, *** - p<0.001

2.6.7.7.O. Repeat-Dose Toxicity Report Title: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog



2.6.7.7.O. TMC278-NC115: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog

Species/strain: dog/Beagle Age at first dose: 8 – 8.5 months Duration of dosing: 182-183 days Duration of postdose: /

Administration route/method: Oral/Gavage Doses: 0 (vehicle), 5, 10, 40 mg/kg/day TMC278 base Test article batch: Vehicle/formulation: PEG400 + CA

Testing facility: J & J PRD. Study no.: TMC278-NC115 GLP compliance: Yes Date of First Dose: - -20

No Observed Adverse Effect Level: / Toxicokinetics:

0 (vehicle) 5 10 40 Dose (mg/kg/day) No. of animals M:0 F:0 M:3 F:3 M:3 F:3 M:3 F:3 Plasma concentration Day 1 (Day of 1st administration)

Cmax ( g/mL) 1.0 0.70 1.3 1.2 1.5 0.82 AUC0-24h ( g.h/mL) 11 9.2 22 20 23 10

Day 86 Cmax ( g/mL) 1.4 1.1 2.0 2.2 2.5 3.1 AUC0-24h ( g.h/mL) 21 18 28 27 41 52

Day 177 Cmax ( g/mL) 1.5 1.4 2.0 1.9 4.0 2.9 AUC0-24h ( g.h/mL) 21 17 26 32 68 43

Tissue concentration (ng/g) Liver

Day 92 10885 7795 22450 15950 50650 40500 Day 183 or 184 10127 7427 13897 12247 25157 25167

Adrenal Day 92 5460 3250 18550 8080 35300 30150 Day 183 or 184 5950 2360 6127 4273 17673 15427

/ = not determined; F = female; M = male. PEG400: Polyethylene glycol 400; CA: citric acid.




Report Title: TMC278-NC115: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 5 10 40 No. of animals M:6 F:6 M:6 F:6 M:6 F:6 M:6 F:6 Dead or Sacrified Moribund 0 0 0 0 0 0 0 0 Clinical Observations Feces, soft 6 6 6 5 6 6 6 6 Salivation - 4 - - - - - 6 Vomiting 6 6 6 6 6 6 6 6 Feces, mucous 4 5 - - - - 5 4 Feces, pale 1 0 - - - - 4 4 Body weight - - - - - - - - Food consumption - - - - - - - - Ophthalmoscopy - - - - - - - - ECG - - - - - - - - Hematology - - - - - - - - Serum Chemistrya 3m Interim kill (n = 6) Cholesterol (mg/dL) 121 158 - - - - 1.397 1.228 Total bilirubin (mg/dL) 0.13 0.13 - - - 1.462 1.308 1.769 Alk. Phosphatase (U/L) 148 101 - 1.842 1.284 1.851 1.723 3.436 6m Terminal kill (n = 4) Cholesterol (mg/dL) 123 161 - - - - 1.699 1.335 Total bilirubin (mg/dL) 0.16 0.12 - 1.333 - 1.667 - 1.667 Alk. Phosphatase (U/L) 224 144 - 1.576 - 1.667 2.304 5.438 Endocrinologya ACTH (pg/mL.h) Day 85 (n = 6) 1159 1582 - 1.139 1.601 1.881 2.323 1.409 Day 176 (n = 4) 583 1051 1.796 - 1.904 - 2.967 - Cortisol (nmol/L.h) Day 85 (n = 6) 1085 1294 0..660 - 0.618 0.488 0.451 0.402 Day 176 (n = 4) 822 966 0.703 - 0.743 0.697 0.485 0.429 a For controls, group means are shown. For treated groups, multiples of control/baseline are shown; - = No noteworthy findings; F = female; M = male.




Report Title: TMC278-NC115: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 5 10 40 Endocrinology (cont’d) Progesterone Day 85 (n = 6) 0.76 57 - 0.965 2.763 0.930 9.474 0.246 Day 176 (n = 4) 1.2 1.4 - 1.214 1.917 2.000 4.333 5.929 17 -OH-progesterone Day 85 (n = 6) 0.56 - 4.643 - 10.357 - 50.000 - Day 176 (n = 4) 0.51 - 4.314 - 5.294 - 21.569 - Organ Weightsa 3m Interim kill (n = 2) Ovaries 1.608 1.297 1.492 1.396 6m Terminal kill (n = 4) Ovaries 1.185 - 1.322 2.030 Gross Pathology 6m Terminal kill No. of animals 4 4 4 4 Ovaries: swollen 0 0 0 3 Uterus: swollen 0 0 0 2 Vagina: swollen 0 0 0 2 a = For controls, group means are shown. For treated groups, multiples of control/baseline are shown; - = No noteworthy findings; F = female; M = male.




Report Title: TMC278-NC115: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog (continued) Noteworthy Findings Dose (mg/kg/day) 0 (vehicle) 5 10 40 Histopathology-3m Interim Kill Adrenal Glands (n° examined) 2 2 2 2 2 2 2 2 Swollen cells/dense staining cytoplasm (zona reticularis)

1 1 2 2

Grade 1 1 1 1 2 Grade 2 1

Swollen cells/dense staining cytoplasm (zona fasciculata)

1 1 1 2

Grade 1 1 1 2 Grade 2 1

Adrenal glands (Oil red O-stain) (n° examined)

2 2 2 2 2 2 2 2

Oil red O-positive droplets (zona fasciculata)

2 2 2 2 2 2 2 2

Grade 2 1 1 Grade 3 1 Grade 4 1 2 1 1 Grade 5 2 1 2 1 1 1

Oil red O-positive droplets (zona reticularis)

2 2 2 2 2 2 2 2

Grade 2 1 2 Grade 3 1 2 1 1 Grade 4 1 1 Grade 5 1 1 2 1 1

F = female; M = male.




TMC278-NC115: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 5 10 40 Histopathology-3m Interim Kill (cont’d)

Epididymides (n° examined) 2 2 2 2 Cellular debris 1 2

Grade 1 1 2 Liver (n° examined) 2 2 2 2 2 2 2 2 Brown pigmented macrophages 1

Grade 1 1 Testes (n° examined) 2 2 2 2 Atrophic tubuli, focal 2

Grade 1 2 Leydig cell hyperplasia/hypertrophy 1 2

Grade 1 1 1 Grade 2 1

Thymus (n° examined) 2 2 2 2 2 2 2 2 Involution 1 1 1 1 2 2 2

Grade 1 1 1 1 1 1 1 Grade 2 1 1 1 Grade 3 1

Ovaries (n° examined) 2 2 2 2 Atretic follicles 2 2 2 2

Grade 2 1 Grade 4 2 Grade 5 1 2 2

Corpora lutea (active) 1 1 1 1 Grade 1 1 1 Grade 2 1 1





Report Title: TMC278-NC115: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 5 10 40 Histopathology-3m Interim Kill (cont’d)

Ovaries (cont’d) Corpora lutea (regressive) 2

Grade 2 1 Grade 5 1

Tertiary follicles 1 2 2 2 Grade 1 1 1 1 Grade 2 1 Grade 3 1 2

Histopathology-6m Terminal Kill Adrenal Gland (n° examined) 4 4 4 4 4 4 4 4 Swollen cells/dense staining cytoplasm (zona reticularis)

1 1 2 2 3 3

Grade 1 1 1 2 2 3 3 Swollen cells/dense staining cytoplasm (zona fasciculata)

1 1 2 2 3 3

Grade 1 1 1 2 2 3 3 F = female; M = male.




Report Title: TMC278-NC115: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 5 10 40 Histopathology-6m Terminal Kill (cont’d)

Adrenal glands (Oil red O-stain) (n° examined)

4 4 4 4 4 4 4 4

Oil red O-positive droplets (zona fasciculata)

4 4 4 4 4 4 4 4

Grade 2 1 Grade 3 1 2 2 3 2 Grade 4 3 1 3 2 2 2 1 1 Grade 5 1 3 1 1

Oil red O-positive droplets (zona reticularis)

4 4 4 4 4 4 4 4

Grade 1 1 Grade 2 2 1 2 4 3 2 Grade 3 1 4 1 3 1 1 1 Grade 4 3 1 1

Epididymides (n° examined) 4 4 4 4 Cellular debris 2 3

Grade 1 1 2 Grade 2 1 1

Reduced amount of spermatozoa 1 1 Grade 1 1 1

Gall bladder (n° examined) 4 4 4 4 4 4 4 4 Prominent bile/brown pigment (epithelial cells)

1 3 3

Grade 1 1 3 3 Liver (n° examined) 4 4 4 4 4 4 4 4 Brown pigmented macrophages 2 2 2

Grade 1 2 2 2 F = female; M = male.




Report Title: TMC278-NC115: Six-Month Repeated Dose Toxicity Study with 3-Month Interim Kill in the Dog (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 5 10 40 Histopathology 6m Terminal Kill (cont’d)

Testes (n° examined) 4 4 4 4 Atrophic tubuli, focal 1 2 4

Grade 1 1 2 4 Leydig cell hyperplasia/hypertrophy 2

Grade 1 2 Ovaries (n° examined) 4 4 4 4 Atretic follicles 4 4 4 4

Grade 2 1 1 Grade 3 2 3 1 Grade 4 1 1 2 Grade 5 2 2

Corpora lutea (regressive) 3 4 3 4 Grade 1 1 2 1 Grade 2 2 2 2 2 Grade 3 2 Grade 5

Tertiary follicles 2 3 2 Grade 1 2 3 2


2.6.7.7.P. Repeat-Dose Toxicity Report Title: Twelve-Month Repeated Dose Oral Toxicity Study in the Dog



2.6.7.7.P. TMC278-NC107: Twelve-Month Repeated Dose Oral Toxicity Study in the Dog

Species/strain: Dog/Beagle Age/weight at first dose: 5 or 6 months/ 6.7 – 9.3 kg for males and 6.3-8.5 kg for females Duration of dosing: 52 weeks Duration of postdose: None

Administration route/method: Oral/gavage Doses: 0, 5, 10 and 40 mg/kg/day TMC278 base Test article batch: Vehicle/formulation: PEG400 + CA

Testing facility: Study No.: TMC278-NC107 GLP compliance: Yes

No Observed Adverse Effect Level: / Toxicokinetics: Dose (mg/kg/day) 0 (vehicle) 5 10 40 No. of animals M:4 F:4 M:4 F:4 M:4 F:4 M:4 F:4 Day 1 Cmax ( g/mL) - - 0.70 0.75 0.90 1.2 2.4 2.5 AUC0-24h ( g.h/mL) - - 11 9.7 15 15 37 41 Day 90 Cmax ( g/mL) - - 1.2 1.4 2.0 2.3 a 3.6 5.5 a AUC0-24h ( g.h/mL) - - 18 21 29 29 a 60 88 a Day 273 Cmax ( g/mL) - - 1.1 1.1 1.3 a 2.3 3.2 3.5 a AUC0-24h ( g.h/mL) - - 16 18 19a 31 60 51 a Day 363 Cmax ( g/mL) - - 1.1 1.5 1.3 b 2.2 4.1 5.5 a AUC0-24h ( g.h/mL) - - 17 19 24 b 36 65 61 a a : n = 3; b : n = 2; / = not determined;.




Report Title: TMC278-NC107: Twelve-Month Repeated Dose Oral Toxicity Study in the Dog (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 5 10 40 No. of animals M:4 F:4 M:4 F:4 M:4 F:4 M:4 F:4 Died or killed prematurely 0 0 0 0 0 0 0 0 Clinical observations Salivation 0 0 1 0 0 1 2 0 Feces liquid 3 4 4 4 4 4 4 4 Body weight gain (kg)c 2.2 3.6 - - - - 0.591 0.444** Food consumption (g) - - - - - - - - Ophthalmoscopy - - - - - - - - ECG - - - - - - - - c For controls, group means are shown. For treated groups, multiples of control/baseline are shown. * - p<0.05, ** - p<0.01, *** - p<0.001. ECG: electrocardiogram; F: female;

M: male; - no noteworthy changes. PEG400: polyethylene glycol; CA: citric acid.




Report Title: TMC278-NC107: Twelve-Month Repeated Dose Oral Toxicity Study in the Dog (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 5 10 40 No. of animals M:4 F:4 M:4 F:4 M:4 F:4 M:4 F:4 Hematology (Week 52)a Red blood cells (M/mm3) 6.96 - - - - - 0.889* - Hemoglobin (g/dL) 16.1 - - - - - 0.869* - Hematocrit (%) 47.3 - - - - - 0.886* - Serum chemistry (Week 52)a Calcium (mg/L) 101 106 - - - - 0.901* 0.934* Inorganic phosphorus (mg/L) 29 - - - - - 1.172 - Creatinine (mg/L) - 7.2 - - - - - 1.181 Alkaline phosphatase (IU/L) 220 288 - - - - 2.877 1.549 Urinalysis (Week 52)a Volume (mL) 65 - - - - - 3.51 - Endocrinology b (Week 52) Progesterone (nmol/L) 0.00 - 1.8 - 3.7 - 7.5 - 17 -OH-progesterone (nmol/L) 1.40 - 6.142 - 8.429 - 14.43 - Estradiol (pmol/L) - - - - - - - - Cortisol (nmol/L) - 27 - - - 0.259 - 0.111 ACTH (pg/mL/h) - - - - - - - - Luteinized hormone (ng/mL) - - - - - - - - Testosterone (nmol/L) - - - - - - - - Organ weights Adrenal glands (g) 1.24 1.29 - - -- 1.233 1.234 1.233 Ovaries (g) - 1.92 - - - 1.396 - 2.193 Gross pathology - - - - - - - - a = For controls, group means are shown. For treated groups, multiples of control/baseline are shown.; b Actual group means are shown as division by zero gives infinity; * - p<0.05, ** - p<0.01, ***. F: female; M: male; - no noteworthy changes




Report Title: TMC278-NC107: Twelve-Month Repeated Dose Oral Toxicity Study in the Dog (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 5 10 40 No. of animals M:4 F:4 M:4 F:4 M:4 F:4 M:4 F:4 Histopathology Adrenal glands Cytoplasmic density (zona fasciculate/reticularis)

0 0 0 4 1 4 3 4

Pigment deposit in the cortex 0 0 0 0 0 0 2 2 Ovaries Antral follicles - 4 - 4 - 4 - 4 Prominent early luteinized follicles - 0 - 0 - 0 - 2 Prominent corpora lutea - 2 - 3 - 3 - 3 Liver Yellow pigmentation 0 0 0 0 0 1 1 3 Kidney Acute interstitial nephritis 0 0 0 0 0 0 2 0 Corticomedullary mineralization 0 0 0 0 0 0 0 3 Testes Hypertrophy of the Leydig cells 0 - 0 - 0 - 2 - F: female; M: male; - no noteworthy changes

2.6.7.7.Q. Repeat-Dose Toxicity

Report Title: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 in the Beagle Dog


A At last measurement. For controls group means are shown. For treated groups, biologically significant or treatment-related differences (%) from comtrols are shown. CONFIDENTIAL Page 135 17AUG2010

2.6.7.7.Q. 96-TOX-4331-004: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 in the Beagle Dog

Species/Strain: Dog, Beagle Duration of Dosing: 28 days Study No: 96-TOX-4331-004 Initial Age: 6 months (approx.) Duration of Post Dose: None Date of First Dose: 19 Method of Administration: Oral gavage Vehicle/Formulation: 50 mM Citric acid GLP Compliance: Yes Special Features: None No observable Effect Level: 3 mg/kg/day 10 mg/kg/day Daily Dose (mg/kg) 0 control 3 10 30 Number of Animals M: 4 F: 4 M: 4 F: 4 M: 4 F: 4 M: 4 F: 4 Toxicokinetics (AUC)ss From Study 96-TOX-4331-004-PK

Day 28 (AUC0- ) g.h/mL - - NC NC NC NC 14.4 24.6 Noteworthy Findings Died or Sacrificed Moribund 0 0 0 0 0 0 0 0 Body Weight (%) A 10.5 kg 8.65 kg -9* 0 0 0 -15** 0 Food Consumption (%) Week 2 385.4g 366.4g 0 0 0 0 0 -18** Week 3 396.4g 386.5g 0 0 0 0 0 -20** Water Consumption Not measured Clinical Observations There were no clinical signs considered treatment-related Ophthalmoscopy There were no treatment related ocular changes Electrocardiography Not measured NC = not calculated * p 0.05 versus control ** p 0.01 versus control

2.6.7.7.Q. Repeat-Dose Toxicity

Report Title: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 in the Beagle Dog



Daily Dose (mg/kg) 0 control 3 10 30 Number of Animals M: 4 F: 4 M: 4 F: 4 M: 4 F: 4 M: 4 F: 4 Hematology Red Blood Cell Counts D D D D Hemoglobin D D D D Hematocrit - - - - D D D D Corpuscular Hemoglobin - - - - - - - D Clinical Chemistry Bilirubin - - - - - I I I Blood Urea Nitrogen - - - - - - I - Creatinine - - - - - - I - Cholesterol - - - - D D D D Urinalysis There were no treatment related effects Organ Weights (%)A There were no significant treatment related effects on organ weights Gross Pathology There were no gross pathological findings that could be attributed to treatment Histopathology Renal Tubular Degeneration and

Regeneration - - - - +4/4 +4/4 ++4/4 ++4/4

Renal Tubular Cell Karyomegaly - - - - +4/4 +4/4 ++4/4 ++4/4 I = Increase D = decrease + = mild ++ = moderate +++ = severe

2.6.7.7.R. Repeat-Dose Toxicity Report Title: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 (bis-POC PMPA; PMPA Prodrug) in the Beagle Dog


NC = Not calculated. A = Initial dose administration on day 2. B = Dosed on days 1, 8, 15 & 22. C= animals moribund, euthanized day 5 or 6 after receiving only one dose. D =At last measurement (day 28), for controls group means are shown. For treated groups % biologically significant or treatment-related differences are shown. A reduced food consumption was however noted for animals in the 60 or 210 mg/kg groups during early stages of the study. E = incidence generally higher in test article groups. F = in surviving animals. G = in moribund animals I = increase, D =decrease. + = Slight to moderate, ++ =moderate to severe CONFIDENTIAL Page 137 17AUG2010

2.6.7.7.R. 98-TOX-4331-003: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 (bis-POC PMPA; PMPA Prodrug) in the Beagle Dog

Species/Strain: Dog, Beagle Duration of Dosing: 28 days Study No: 98-TOX-4331-003 Initial Age: 6–7 months Duration of Post Dose: 1 day Date of First Dose: 19 Method of Administration: Oral gavage Vehicle/Formulation: 50 mM citric acid GLP Compliance: Yes Special Features: None No Observable Effect Level: <30 mg/kg/day Daily Dose (mg/kg) 0 (Control) bid 30 qd 15 bid 60 q2dA 210 q7d B Number of Animals M: 4 M: 4 M: 4 M: 4 M: 4 Toxicokinetics (AUC)ss From Study 98-TOX-4331-003-PK

AUC0- ( g.h/mL) Day 28 - NC 29.4 67.0 219 Noteworthy Findings Died or Sacrificed Moribund 0 0 0 0 2C Body weight (%) d 10.32 kg 0 0 0 0 Food Consumption d 317.3g/animal 0 0 0 0 Water Consumption Not measured Clinical Observations Vomitus, Abnormal feces

(mucoid, soft liquid) E 3/4 3/4 4/4 4/4 4/4

Thin, Dehydrated - 1/4 - 2/4 2/4 Salivation - 1/4 - 3/4 2/4 Cold to Touch - - - 1/4 3/4 Decreased Activity - - - 1/4 2/4 Ophthalmoscopy Not measured Electrocardiography Not measured

2.6.7.7.R. Repeat-Dose Toxicity Report Title: A 28-Day Oral Gavage Toxicity Study of GS-4331-05 (bis-POC PMPA; PMPA Prodrug) in the Beagle Dog



Daily Dose (mg/kg) 0 (Control) bid 30 qd 15 bid 60 q2dA 210 q7d B Number of Animals M: 4 M: 4 M: 4 M: 4 M: 4 Hematology There were no changes in hematology parameters that were considered treatment-related. Increases in WBC, RBC,

hemoglobin & hematocrit values in moribund dogs in the 210mg/kg group were considered due to dehydration & the agonal condition of the animals.

Clinical Chemistry Cholesterol - D D D IG Creatinine - I I I IF IG Creatinine Kinase - - - - IG BUN - - - - IG Total Bilirubin - I I I IF IG Total Protein - - - - IG Potassium - - - - IG Calcium - - - - IG Phosphorous - - - - IG Triglycerides - I I I - AST - - - - IG ALT - - - - IG Albumin - - - - IG Globulin - - - - IG Glucose - - - - DG Sodium - - - - DG Chloride - - - - DG Bicarbonate - - - - DG Urinalysis Urine Volume - I I I IF Urine pH - - - - IG Urine Protein - - - - DG Organ Weights (absolute/ relative) - Thymus - D4/4 D4/4 D3/4 D4/4

2.6.7.7.S. Repeat-Dose Toxicity Report Title: A 13- and 42-Week Oral Gavage Toxicity Study (with 13 Week Recovery Period) of Bis-POC PMPA (GS-4331-05) in the Beagle Dog


NC not calculated due to insufficient data. A= At last measurement. For controls group means are shown. For treated groups, biologically significant or treatment-related differences (%) from controls are shown. Body weight loss was observed in the 10 & 30 mg/kg groups during 1st 2 weeks of dosing, subsequent gains were similar to control throughout remainder of dosing period. B= Mixed male and female value. C Animal sacrificed on day 84 because of deterioration in condition most likely as a consequence of a gavage accident. * P < 0.01, ** p < 0.05, + = mild, ++ moderate, +++ severe


2.6.7.7.S. 97-TOX-4331-001: A 13- and 42-Week Oral Gavage Toxicity Study (with 13-Week Recovery Period) of Bis-POC PMPA (GS-4331-05) in the Beagle Dog

Species/Strain: Dog, Beagle) Duration of Dosing: 13 or 42 weeks Study No: 97-TOX-4331-001 Initial Age: 6–7 months (approx.) Duration of Post Dose: 13 weeks Date of First Dose: 19 (M and F respectively)

Method of Administration: Oral gavage

Vehicle/Formulation: 50 mM Citric acid GLP Compliance: Yes Special Features: None No observable Effect Level: < 3 mg/kg/day Daily Dose (mg/kg) 0 control 3 10 30 Number of Animals (including 2 recovery animals per sex per group) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Toxicokinetics (AUC)ss From Study 97-TOX-4331-001-PK

Week 13 (AUC0- ) g.hr/mL - - NC - 5.71 - 28.1B 28.1B Week 26 (AUC0- ) g.hr/mL - - NC - 5.83 - 28.5B 28.5B Week 42 (AUC0- ) g.hr/mL - - NC NC 6.84B 6.84B 31.1B 31.1B Noteworthy Findings Died or Sacrificed Moribund 1C 0 0 0 0 0 0 0 Body Weight (%) A 13.67 kg 11.48 kg 0 0 0 0 0 0 Food Consumption (%) There were no treatment related effects on food consumption Water Consumption Not measured Clinical Observations There were no treatment-related clinical signs observed Ophthalmoscopy There were no treatment related ophthalmological changes Electrocardiography There was no electrocardiographic evidence of test article action throughout the study.





Daily Dose (mg/kg) 0 control 3 10 30 Number of Animals M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Hematology APTT - - - - - - I I Clinical Chemistry Cholesterol - - D D D D D D Chloride - - - I - I I I Creatinine - - - - - I I I Potassium - - - - - - D D Bilirubin - - - - - - I I Alkaline phosphatase - - - - - - I I Creatine kinase - - - - - - I I Bone 1,25-dihydroxy-Vitamin D3 - - - - - - D D N-telopeptide - - - - - - I I + Bone Mineral Content/Density - - - - - - D D Urinalysis Glucosuria - - - - - - I I Proteinuria - - - - - - I I Urinary Output - - - - - - I I Urinary Calcium - - - - - - I I Organ Weights (%)A Kidney 4.642g 4.462 0 0 0 0 +27* 0 Liver (absolute) 449.97g 286.45g 0 0 0 0 -34** 0 Gross Pathology Pale Discoloration of Renal Cortex - - - - 1/4 - 4/4 4/4





Daily Dose (mg/kg) 0 control 3 10 30 Number of Animals M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Histopathology Interstitial Nephritis - - - - - - 2/4 + Renal Tubular Karyomegaly - - 1/4 1/4 + ++Renal Tubular Karyomegaly 4/4 4/4 4/4 4/4 + Tubular Dilatation - - - - 1/4 - 4/4 4/4 + Tubular Degeneration/Regeneration - - - - - 1/4 4/4 4/4 + Centrolobular Sinusoidal Cell

Pigment Accumulation - - - - - - 4/4 3/4

+ Centrolobular Sinusoidal Dilatation/Congestion

- - - - - - 4/4 3/4

Post Dose Evaluation: There was incomplete resolution of abnormalities in clinical and microscopic pathology parameters at the end of the recovery period (week 55/54) in dogs in the 10 and/or 30 mg/kg groups but complete resolution in the 3 mg/kg group. There was also incomplete resolution of bone alterations at the end of the recovery period (see below)

Clinical Chemistry Alkaline Phosphatase - - - - - - I I Bilirubin - - - - - - I Chloride - - - - - I I I Bone 1,25-dihydroxy-Vitamin D3 - - - - - - - D N-telopeptide - - - - - - I I Bone Mineral Content - - - - - - D D Urinalysis Glucosuria - - - - - - I I Gross pathology Pale discoloration of renal cortex - - - - 2/2 - 1/2 2/2





Daily Dose (mg/kg) 0 control 3 10 30 Number of Animals M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Histopathology Interstitial Nephritis - - - - 2/2 1/2 - 2/2 + Tubular Dilatation - - - - - - 1/2 - + Renal Tubular Karyomegaly - - - - 2/2 2/2 2/2 2/2 + Tubular Degeneration/Regeneration - - - - 2/2 2/2 - 2/2

2.6.7.7.T. Repeat-Dose Toxicity Report Title: 4-Week Oral Gavage Toxicity and Toxicokinetic Study with Emtricitabine/Tenofovir Disoproxil Fumarate (FTC/TDF) in Male Dogs with a 4-Week Recovery Period

Test Article: Emtricitabine and Tenofovir Disoproxil Fumarate (alone and in combination)


2.6.7.7.T. TX-164-2004: 4-Week Oral Gavage Toxicity and Toxicokinetic Study with Emtricitabine/Tenofovir Disoporxil Fumarate (FTC/TDF) in Male Dogs with a 4-Week Recovery Period

Species/Strain: Dog/Beagle Duration of Dosing: 4 weeks (29 days) Study Number: Gilead Sciences, Study No. TX-164-2004, Covance 6511-155

Initial Age: Approximately 4.5 to 5 months old Duration of Postdose: 4 weeks Date of First Dose: 20 Method of Administration: Oral gavage Vehicle/Formulation: 50mM citrate buffer GLP Compliance: Yes Special Features: Immunophenotyping and Natural Killer Cell Assay; males only were tested. No observable Effect Level: 20 mg/kg/day (FTC), 2/3 mg/kg/day (FTC/TDF)

Daily Dose (mg/kg) 0 (Control) 20 Emtricitabine

(FTC) 30 Tenofovir Disoproxil

Fumarate (TDF) 2 FTC/3 TDF 20 FTC/ 30 TDF Number of Animals M: 4 M: 4 M: 4 M: 4 M: 6a Toxicokinetics:

AUCtau (Day 27) (μg.h/mL for FTC) (ng.h/mL for TFV)

- 25.3 18413 TFV

2.7 FTC 909 TFV

32.3 FTC 20872 TFV

Cmax (Day 27) (μg/mL for FTC) (ng/mL for TFV)

- 6.9 2996 TFV

0.9 FTC 154 TFV

7.3 FTC 3439 TFV

Noteworthy Findings

Died or Sacrificed Moribund 0 0 0 0 0 Body Weight (%) - - - - - Food Consumption (%) - - - - -

- = No noteworthy findings. + Mild ++ Moderate +++ Marked TFV = Tenofovir a Two males designated for recovery. (Continued)

2.6.7.7.T. Repeat-Dose Toxicity Report Title: 4-Week Oral Gavage Toxicity and Toxicokinetic Study with Emtricitabine/Tenofovir Disoproxil Fumarate (FTC/TDF) in Male Dogs with a 4-Week Recovery Period

Test Article: Emtricitabine and Tenofovir Disoproxil Fumarate (alone and in combination)


Daily Dose (mg/kg) 0 (Control) 20 Emtricitabine

(FTC) 30 Tenofovir Disoproxil

Fumarate (TDF) 2 FTC/3 TDF 20 FTC/ 30 TDF Number of Animals M: 4 M: 4 M: 4 M: 4 M: 6a Water Consumption Not measured Clinical Observations There were no treatment-related clinical signs observed. Ophthalmoscopy There were no treatment-related clinical signs observed. Electrocardiography Not measured Hematology Increase APTT - - + - + Increase MCV - + - - + Serum Chemistry Creatinine - - I - I Urinalysis - - - - - Organ Weights (%) - - - - - Gross Pathology - - - - - Histopathology - - - - -

+ Tubular epithelial necrosis - - 2/4 3/4 + Tubular regeneration 1/4 1/4 4/4 - 4/4

Additional Examinations Immunophenotyping - - - - - NK cell analysis - - - - -

Postdose Evaluation: Number Evaluated - - - - 2 Findings - - - - -

- = No noteworthy findings. I = Increased. a Two males designated for recovery. The hematological and kidney microscopic effects were reversed following recovery.

2.6.7.7.U. Repeat-Dose Toxicity Report Title: A 30-day Oral Toxicity Study in Cynomolgus Monkeys Given 524W91



2.6.7.7.U. TOX600: Repeat-Dose Toxicity: A 30-Day Oral Toxicity Study in Cynomolgus Monkeys Given 524W91

Species/Strain: Monkey / Cynomolgus (Macaca fascicularis)

Duration of Dosing: At least 28 days Study Number: TOX600

Initial Age: Approximately 2.5–7 year males and 6–17 year females

Duration of Postdose: 3 weeks

Date of First Dose: 19 Method of Administration: Oral, divided doses spaced by approximately six hours

Vehicle/Formulation: Vehicle control: 0.5% aqueous methylcellulose

GLP Compliance: Yes

No Observed Adverse-Effect Level: A NOEL of 400 mg/kg/day was established.

Daily Dose (mg/kg) 0 (Control) 80 400 2000 Number of Animals M: 5 F: 5 M: 5 F: 5 M: 5 F: 5 M: 5 F: 5 Toxicokinetics: AUCA

0-6 ( g.hr/mL) Day 3 N/A N/A 37.1 37.8 170 170 631 765 Day 27 N/A N/A 31.6 33.6 196 164 730 732 Cmax ( g/mL) Day 3 N/A N/A 14.6 15.8 58.3 66.0 194 204 Day 27 N/A N/A 12.4 12.9 63.8 63.0 202 191 Noteworthy Findings

Died or Sacrificed Moribund 0 0 0 0 0 0 0 0 Body Weight (%) No treatment related changes Food Consumption (%) No treatment related changes Water Consumption Not measured Clinical Observations Increased incidence of soft feces in females given 2000 mg/kg/day

A Drug plasma concentrations – No gender-specific differences were noted in any of the pharmacokinetic parameters measured. Also, no significant differences in pharmacokinetic parameters were determined between Days 3 and Day 27. Cmax and the AUC values increased in proportion to the dose. No significant drug accumulation occurred over the dosing period.

N/A = not analyzed

2.6.7.7.U. Repeat-Dose Toxicity Report Title: A 30-day Oral Toxicity Study in Cynomolgus Monkeys Given 524W91



Daily Dose (mg/kg) 0 (Control) 80 400 2000 Number of Animals M: 5 F: 5 M: 5 F: 5 M: 5 F: 5 M: 5 F: 5 OphthalmoscopyB No treatment related changes ElectrocardiographyC No treatment related changes Hematology No treatment related changes Serum Chemistry No treatment related changes Urinalysis No treatment related changes Organ Weights (%)D No treatment related changes Gross Pathology Necropsy D No treatment related changes Histopathology D No treatment related changes Additional Examinations Electronic microscopyE Focal areas of "moth-eaten" mitochondria were seen in control (Group 1) cardiac muscle specimens. Moth-eaten

mitochondria were also seen in cardiac muscle in the 2000 mg/kg/day group (Group 4) but some of the Group 4 samples exhibited normal mitochondria. Since no drug-related ultrastructural changes were seen in cardiac muscle in a 90 day study in monkeys given emtricitabine, these mitochondrial findings were not considered to be drug-related. No drug-related electron microscopic changes were observed in skeletal muscle at any dose level.

CSF concentration 4.3% of plasma levels for 80 mg/kg/day; 3.7% of plasma levels for 400 mg/kg/day; 3.7% of plasma levels for 2000 mg/kg/day groups. CSF levels increased as plasma levels increased.

Neurophysiology No treatment related changes Neuropathology No treatment related changes Postdose Evaluation: 2 monkeys/sex/group were held for a 3 week post-dose drug-free recovery period

B Ophthalmic examination (pupillary dilation was achieved with 1% tropicamide ophthalmic solution and ketamine HCl for immobilization). C Electrocardiography (unanesthetized monkeys) D At the termination of dosing 3 monkeys/sex/group were sacrificed, examined grossly, selected organs were weighed, and tissues were taken for histopathological examination. E Electronic microscopy – samples of skeletal and cardiac muscle from the control and 2000 mg/kg/day group were taken for ultrastructural examination.

2.6.7.7.V. Repeat-Dose Toxicity Report Title: A 3-Month Oral Toxicity Study in Cynomolgus Monkeys Given 524W91



2.6.7.7.V. TOX627: Repeat-Dose Toxicity: A 3-Month Oral Toxicity Study in Cynomolgus Monkeys Given 524W91

Species/Strain: Monkey/Cynomolgus (Macaca fascicularis)

Duration of Dosing: 3 months Study Number: TOX627

Initial Age: Approximately 2–5.5 years Duration of Postdose: 3 weeks Date of First Dose: 19 Method of Administration: Oral, divided doses

spaced by approximately six hours


GLP Compliance: Yes

No Observed Adverse-Effect Level: 1000 mg/kg


AUC 0-6 ( g.h/mL)A Day 3 N/A N/A 12.2 13.4 71.0 51.9 331 233 Day 87 N/A N/A 13.1 14.5 64.8 54.6 403 274

Cmax ( g/mL) Day 3 N/A N/A 5.19 6.06 28.9 22.9 101 92.5 Day 87 N/A N/A 5.27 5.96 26.0 23.1 117 94.4 Noteworthy Findings

Died or Sacrified Moribund 0 0 0 0 0 0 0 0 Body Weight (%) No treatment related changes Food Consumption (%) No treatment related changes Water Consumption Not measured Clinical Observations No treatment related changes OphthalmoscopyB No treatment related changes ElectrocardiographyC No treatment related changes

N/A = Not Applicable A Drug plasma concentrations – Cmax and AUC increased with the dose and there was no evidence of accumulation: Systemic exposure to emtricitabine was similar between

sexes and between days 3 and 87. B Ophthalmic examination (pupillary dilation was achieved with 1% tropicamide ophthalmic solution and ketamine HCl for immobilization). C Electrocardiography (unanesthesized monkeys)

2.6.7.7.V. Repeat-Dose Toxicity Report Title: A 3-Month Oral Toxicity Study in Cynomolgus Monkeys Given 524W91



Daily Dose (mg/kg) 0 (Control) 40 200 1000 Number of Animals M: 5 F: 5 M: 5 F: 5 M: 5 F: 5 M: 5 F: 5 Hematology No treatment related changes Serum Chemistry No treatment related changes Urinalysis No treatment related changes Organ Weights (%) No treatment related changes Gross Pathology Necropsy No treatment related changes Histopathology No treatment related changes Additional Examinations Neurophysiology No treatment related changes Neuropathology No treatment related changes Electronmicroscopy No treatment related changes Postdose Evaluation 2 monkeys/sex/group were held for a 3 week post-dose drug-free recovery period

D At the termination of dosing 3 monkeys/sex/group were sacrified, examined grossly, selected organs were weighed and tissues were taken for histopathologic examination. E Electronmiscroscopy – samples of liver, skeletal and cardiac muscle for 3 monkeys/sex/group were taken for ultrastructural examination.

2.6.7.7.W. Repeat-Dose Toxicity Report Title: 52-Week Oral Toxicity Study of TP-0006 (Emtricitabine) in Cynomolgus Monkeys with a 4-Week Recovery Period



2.6.7.7.W. TOX032: Repeat-Dose Toxicity: 52-Week Oral Toxicity Study of TP-0006 (Emtricitabine) in Cynomolgus Monkeys with a 4-Week Recovery Period

Species/Strain: Monkey / Cynomolgus (Macaca fascicularis)

Duration of Dosing: 52 weeks Study Number: TOX032

Initial Age: Approx. 3–7 years Duration of Postdose: 4 weeks Date of First Dose: 19 Method of Administration: Oral, divided doses

spaced by approximately six hours


GLP Compliance: Yes

Special Features: Neurophysiology No Observed Adverse-Effect Level: No observed effect level (NOEL) – 200 mg/kg/day


AUC0-24 ( g.h/mL)A Week 13 N/A N/A 16.1 18.1 74.9 100 266 196 Week 26 N/A N/A 17.0 22.9 103 120 342 321 Week 52 N/A N/A 18.1 33.3 97.2 97.8 274 238 Cmax ( g/mL) Week 13 N/A N/A 3.7 2.9 11.1 16.9 41.5 26.9 Week 26 N/A N/A 3.7 4.8 15.6 24.8 51.2 39.3 Week 52 N/A N/A 4.3 7.3 17.4 21.3 50.6 37.6 T1/2 (hr) Week 13 N/A N/A 2.70 2.76 4.42 1.60 2.46 3.00

Week 26 N/A N/A 2.49 2.57 2.16 1.67 1.94 2.91 Week 52 N/A N/A 2.19 1.57 1.84 1.49 1.99 1.76

A Emtricitabine was rapidly and well absorbed with peak plasma concentrations occurring between 0.5 to 2 hours post dosing. Plasma half-life was 2–4 hours for all dose levels. Half-life may have been underestimated because of the short (6 hour) sampling period. A longer half-life was expected, based on the measurable pre-dose drug concentrations and the slightly higher plasma emtricitabine concentrations after repeated dosing. No major differences in plasma emtricitabine exposure between male and female monkeys. Plasma Cmax, AUC0-6, and estimated steady-state AUC0-24 (Weeks 13, 26, and 52) increased linearly over the dose range of 50 to 500 mg/kg/day.

2.6.7.7.W. Repeat-Dose Toxicity Report Title: 52-Week Oral Toxicity Study of TP-0006 (Emtricitabine) in Cynomolgus Monkeys with a 4-Week Recovery Period




Noteworthy Findings Died or Sacrificed Moribund 0 0 0 0 0 0 0 0 Body Weight (%) No treatment related changes. Food Consumption (%) Not reported Water Consumption Not measured Clinical Observations No treatment related changes Ophthalmoscopy No treatment related changes Electrocardiography No treatment related changes Hematology Slightly decreased mean erythrocyte counts, increased mean corpuscular volume and increased mean corpuscular

hemoglobin in females treated with 500 mg/kg/day Serum Chemistry The activity of alanine aminotransferase was increased in several individuals (including controls). No treatment related

patterns were identified Urinalysis No treatment related changes Organ Weights (%) No treatment related changes Gross Pathology Necropsy No treatment related changes Histopathology No treatment related changes Additional Examinations

Neurophysiology No treatment related changes Postdose Evaluation: 2 monkeys/sex/group were held for a 4 week post-dose drug-free recovery period

2.6.7.7.X. Repeat-Dose Toxicity Report Title: Endocrinological Oral (Gavage) 8-Week Study in the Female Sexually Immature Cynomolgus Monkey



2.6.7.7.X. TMC278-NC248: Endocrinological Oral (Gavage) 8-week Study in the Female Sexually Immature Cynomolgus Monkey

Species/Strain: Monkey/cynomolgus Initial Age/weight: 1-2 years/1.5-2.0 kg Duration of dosing: 8 weeks

Administration route/method: Oral/Gavage Doses: 0 (vehicle), 100 , 250 mg base eq./kg/day TMC278.HCl twice daily Test article batch: Vehicle/formulation: 1% HPMC + 0.5% Tween 20

Testing facility: Study No.: TMC278-NC248 GLP compliance: Yes Date start of study: 20

Special features: Hormone analysis No Observed Adverse Effect Level: / Toxicokinetics: Dose (mg base eq./kg/day) 0 (vehicle) a 200 500 No. of animals F:8 F:8 F:8 Day 55 Cmax 1 ( g/mL) / 0.14 0.31 Cmax 2 ( g/mL) 0.18 0.31 AUC0-24h ( g.h/mL) / 2.7 4.6




Report Title: TMC278-NC248: Endocrinological Oral (Gavage) 8-week Study in the Female Sexually Immature Cynomolgus Monkey (continued) Noteworthy Findings Dose (mg base eq./kg/day) 0 200 500 N° of animals F:8 F:8 F:8 Died or killed prematurely 0 0 0 Clinical observations - - - Body weights (kg) - - - Menses - - - ACTH (pmol/L) - - - Cortisol (nmol/L) - - - 17-hydroxyprogesterone (nmol/L) D 15 7.57 11.7 13.0 17-hydroxyprogesterone (nmol/L) D 29 7.97 17.0 18.2 17-hydroxyprogesterone (nmol/L) D 43 6.49 16.1 14.9 Progesterone (nmol/L) D15 10.6 11.3 14.5 Progesterone (nmol/L) D29 9.65 14.3 16.2 Progesterone (nmol/L) D43 8.12 13.4** 14.3** a In the control-dosed group, plasma concentrations (26 out of 80) were above the limit of quantification (0.001 g/mL) and ranged from 0.001 to 0.003 g/mL. ** p 0.01 (Analysis of variance); D: day; / = not determined; - = no noteworthy findings; HPMC: hydroxypropylmethylcellulose; F: female




Report Title: TMC278-NC248: Endocrinological Oral (Gavage) 8-week Study in the Female Sexually Immature Cynomolgus Monkey (continued)

Noteworthy Findings: Dose (mg base eq./kg/day) 0 200 500 N° of animals F:8 F:8 F:8 Androstenedione (nmol/L) D15 2.64 1.94* 1.81* Androstenedione (nmol/L) D29 2.10 1.66 1.53 Androstenedione (nmol/L) D43 2.01 1.44 1.27 DHEA (nmol/L) D15 245 264 196 DHEA (nmol/L) D29 289 296 225 DHEA (nmol/L) D43 257 270 199 Estradiol - - - LH - - - Hematology - - - Clinical chemistry - - - Urine analysis - - - Organ weights - - - Macroscopic - - - Histopathology Thyroid Follicular cell hypertrophy 1 3 4 * p 0.05 (Analysis of variance); D: day; - no noteworthy findings; F: female

2.6.7.8.A. Genotoxicity: In Vitro Report Title: Mutagenicity Test with FTC in the Salmonella – Escherichia Coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay



2.6.7.8. Genotoxicity: In Vitro 2.6.7.8.A. 18637-0-409R: Mutagenicity Test with FTC in the Salmonella – Escherichia Coli/Mammalian-Microsome Reverse

Mutation Assay with a Confirmatory Assay

Test for Induction of: Reverse mutations at the histidine locus in S. typhimurium & Reverse mutations at the tryptophan locus in E. coli

No. of Independent Assays: a) - S9: 4 experiments b) + S9: 4 experiments

Study No: 18637-0-409R

Strains: Salmonella typhimurium (TA98, TA100, TA1535, TA1537) & Escherichia coli (WP2uvrA)

No. of Replicate Cultures: A Rangefinding study a) single b) single Mutagenicity assays a) triplicate b) triplicate

Metabolizing System: Aroclor™-induced rat liver (S9 mix) No. of Cells Analyzed/Culture: 0.5 x 109 bacteria/mL

Vehicles: DMSO For Test Articles: FTC- lot ( S9)

For Positive Controls: DMSO ( S9) 2-aminoanthracene or 2-nitrofluorene or sodium azide or ICR-191 or 4-nitroquinoline-N-oxide

GLP Compliance: Yes

TreatmentB: After the overlay had solidified, the plates were inverted and incubated for 48 8 hr at 37 2°C. Date of Treatment: , 19 – , 19

Cytotoxic Effects: No cytotoxicity was observed in the presence or absence of S9 mix as evidenced by a normal background lawn and no test article precipitation.

Genotoxic Effects: Range-finding study – No decrease in the number of revertants per plate. Mutagenicity assays – No positive increases in the number of revertants per plate were observed In the confirmatory assay - No positive increases in the number of revertants per plate were observed.

A Dose Range-finding study: a) - S9: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330, 5000 g/plate. Vehicle control. b) + S9: (same) Mutagenicity assays (initial, confirmatory, additional): a) - S9: 100, 333, 1000, 3330, 5000 g/plate. Positive and vehicle controls. b) + S9: (same)

B When all the required components have been added (tester strain, vehicle or test article, ± S9), the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 100 mm petri dish.




Genotoxic Effects: (Cont’d)

The 2.1-fold, non-dose responsive increase was considered artifactual due to a lower than routinely observed vehicle control value. Effects of the positive control: All positive and negative controls in the initial, confirmatory, and additional assay were within the historical control ranges. The exception was in the confirmatory assay; the mean vehicle control value for tester strain TA98 (-S9 mix) was lower than the acceptable range. The tester strain was retested (-S9 mix) and all data were acceptable.The positive control articles produced acceptable responses and met study criteria for positive response. Conclusion: Emtricitabine did not cause a positive increase in the number of revertants per plate of any of the tester strains in either the presence or absence of microsomal enzymes prepared from Arocolor®-induced rat liver (S9).




Study No.: 18637-0-409R Continued Assay 1: Experiment ID 18637-B1 Metabolic Dose Level Mean revertants per plate with standard deviation Activation ( g/plate) TA98 TA100 TA1535

MEAN S.D. MEAN S.D. MEAN S.D. Microsomes: rat liver Vehicle Control 28 7 114 11 13 4

Test Article 100 27 5 116 10 10 2 333 28 11 83 7 10 6 1000 26 6 100 15 10 3 3330 23 5 93 12 15 3 5000 21 7 85 9 12 2 Positive Control** 1116 109 1103 128 133 8 Microsome: none Vehicle Control 10 4 87 8 11 5

Test Article 100 14 4 93 8 13 2 333 14 3 88 9 12 1 1000 12 2 90 5 13 3 3330 11 1 73 9 12 2 5000 12 4 74 16 11 3 Positive Control*** 142 30 542 14 527 82

** TA98 2-aminoanthracene: 2.5 g/plate *** TA98 2-nitrofluorene: 1.0 g/plate TA100 2-aminoanthracene: 2.5 g/plate TA100 sodium azide: 2.0 g/plate TA1535 2-aminoanthracene: 2.5 g/plate TA1535 sodium azid: 2.0 g/plate TA1537 2-aminoanthracene: 2.5 g/plate TA1537 ICR-191: 2.0 g/plate WP2uvrA 2-aminoanthracene: 25.0 g/plate WP2uvrA 4-nitroquinoline-N-oxide: 1.0 g/plate




Assay 1: Experiment ID 18637-B1 (Continued) Metabolic Dose Level Mean revertants per plate with standard deviation Activation ( g/plate) TA1537 WP2uvrA MEAN S.D. MEAN S.D. Microsomes: rat liver Vehicle Control 12 2 13 5

Test Article 100 7 3 15 1 333 8 2 8 2 1000 8 2 9 2 3330 12 2 11 4 5000 7 2 12 1 Positive Control** 150 21 160 28 Microsome: none Vehicle Control 6 1 8 4

Test Article 100 9 4 14 5 333 6 1 9 2 1000 7 2 11 4 3330 3 3 9 1 5000 5 2 7 3 Positive Control*** 289 30 253 10

** TA98 2-aminoanthracene 2.5 g/plate *** TA98 2-nitrofluorene 1.0 g/plate TA100 2-aminoanthracene 2.5 g/plate TA100 sodium azide 2.0 g/plate TA1535 2-aminoanthracene 2.5 g/plate TA1535 sodium azide 2.0 g/plate TA1537 2-aminoanthracene 2.5 g/plate TA1537 ICR-191 2.0 g/plate WP2uvrA 2-aminoanthracene 25.0 g/plate WP2uvrA 4-nitroquinoline-N-oxide 1.0 g/plate




Assay 2: Experiment ID 18637-C1 Metabolic Dose Level Mean revertants per plate with standard deviation Activation ( g/plate) TA98 TA100 TA1535

MEAN S.D. MEAN S.D. MEAN S.D. Microsomes: rat liver Vehicle Control 16 5 97 7 11 5

Test Article 100 17 5 94 8 10 1 333 10 4 93 18 10 1 1000 12 3 67 5 11 3 3330 15 6 70 16 11 3 5000 11 2 52 9 13 5 Positive Control** 1045 44 1051 37 109 6 Microsome: none Vehicle Control 6* 2 69 9 5 1

Test Article 100 NC - 66 7 10 1 333 NC - 50 5 8 5 1000 NC - 79 13 8 2 3330 NC - 57 7 5 3 5000 NC - 56 7 2 3 Positive Control*** NC - 411 46 431 38

** TA98 2-aminoanthracene: 2.5 g/plate *** TA98 2-nitrofluorene: 1.0 g/plate TA100 2-aminoanthracene: 2.5 g/plate TA100 sodium azide: 2.0 g/plate TA1535 2-aminoanthracene: 2.5 g/plate TA1535 sodium azide: 2.0 g/plate TA1537 2-aminoanthracene: 2.5 g/plate TA1537 ICR-191: 2.0 g/plate WP2uvrA 2-aminoanthracene: 25.0 g/plate WP2uvrA 4-nitroquinoline-N-oxide: 1.0 g/plate

* The mean vehicle control value for tester strain TA98 in the absence of S9 was not within the acceptable range. See retest data (experiment ID 18637-D1)




Assay 2: Experiment ID 18637-C1 (Continued)

Metabolic Dose Level Mean revertants per plate with standard deviation Activation ( g/plate) TA1537 WP2uvrA MEAN S.D. MEAN S.D. Microsomes: rat liver Vehicle Control 9 1 7 4

Test Article 100 7 4 12 4 333 8 3 10 4 1000 5 0 10 1 3330 7 2 15 4 5000 6 4 12 3 Positive Control** 165 27 268 27 Microsome: none Vehicle Control 6 1 11 3

Test Article 100 6 1 10 6 333 5 1 12 5 1000 4 2 12 3 3330 2 2 9 4 5000 3 1 14 9 Positive Control*** 236 34 203 35

** TA98 2-aminoanthracene: 2.5 g/plate *** TA98 2-nitrofluorene: 1.0 g/plate TA100 2-aminoanthracene: 2.5 g/plate TA100 sodium azide: 2.0 g/plate TA1535 2-aminoanthracene: 2.5 g/plate TA1535 sodium azide: 2.0 g/plate TA1537 2-aminoanthracene: 2.5 g/plate TA1537 ICR-191: 2.0 g/plate WP2uvrA 2-aminoanthracene: 25.0 g/plate WP2uvrA 4-nitroquinoline-N-oxide: 1.0 g/plate




Assay 3: Experiment ID 18637-D1 Metabolic Dose Level Mean revertants per plate with standard deviation Activation ( g/plate) WP2uvrA WP2uvrA

1 2 3 MEAN S.D. Microsomes: rat liver Vehicle Control 18 24 7 16 9 Test Article 100 11 14 13 13 2 333 15 12 15 14 2 1000 9 12 17 13 4 3330 13 8 12 11 3 5000 13 10 9 11 2 Positive Control** 289 227 182 233 54

Metabolic Activation

Dose Level ( g/plate) TA98 TA98

1 2 3 MEAN S.D. Microsomes: rat liver Vehicle Control 14 15 15 15 1 Test Article 100 10 12 11 11 1 333 17 22 22 20 3 1000 6 17 9 11 6 3330 9 7 12 9 3 5000 6 7 11 8 3 Positive Control*** 137 129 141 136 6

** WP2uvrA 2-aminoanthracene: 25.0 g/plate *** TA98 2-nitrofluorene: 1.0 g/plate

2.6.7.8.B. Genotoxicity: In Vitro Report Title: MUT203: Salmonella/Mammalian-Microsome Assays with 524W91 (Ames Test)



2.6.7.8.B. MUT203: Salmonella/Mammalian-Microsome Assays with 524W91 (Ames Test)

Test for Induction of: Reverse mutations at histidine locus in S. typhimurium

No. of Independent Assays: a) - S9: 3 experiments b) + S9: 3 experiments

Study No: MUT203

Strains: Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538)

No. of Replicate Cultures: A Rangefinding study a) single b) single Mutagenicity assays a) triplicate b) triplicate

Metabolizing System: Aroclor™-induced rat liver (S9 mix) No. of Cells Analyzed/Culture: 0.5 x 109 bacteria/mL

Vehicles For Test Articles: DMSO 524W91UO reference number 92/0277-049-C and (± S9)

Vehicles For Positive Controls: DMSO + 2-aminoanthracene or 2-nitrofluorene or Sodium azide or ICR-191 or 2-nitrofluorene and (± S9)

GLP Compliance: Yes

Treatment: B Incubation was at 37 ± 2°C for 48 ± 8 hours. Date of Treatment: , 19 – , 19

Cytotoxic Effects: No cytotoxicity was observed in presence or absence of S9 mix as evidenced by a normal background lawn and no test article

precipitation. Genotoxic Effects: Emtricitabine exerted no detectable mutagenic activity in the Ames Salmonella assay, either by plate incorporation or by suspension

preincubation modification.

A Dose Range finding study: a) - S9: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330, 5000 g/plate. Vehicle control. b) + S9: (same)

Mutagenical, confirmatory, additional: a) - S9: 100, 333, 1000, 3330, 5000 g/plate. Positive and vehicle controls. b) +S9: (same) B The test article, the tester strain, and the S9 mix (where appropriate) were combined in molten agar which was overlaid into a minimal agar.




Assay 1: Experiment ID 15437-B1

MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION Metabolic Activation

Dose Level ( g/plate) TA98 TA100 TA1535

MEAN S.D. MEAN S.D. MEAN S.D. Microsomes: rat liver Vehicle Control 35 5 251 9 13 1 Test Article 100 28 7 NC - 14 5 333 35 3 NC - 13 2 667 30 6 NC - 13 3 1000 23 7 NC - 10 1 3330 40 9 NC - 8 3 5000 28 11 NC - 14 8 Positive Control** 976 56 1182 59 181 17 Microsome: none Vehicle Control 19 8 159 26 12 4 Test article 100 16 3 167 13 11 3 333 16 4 165 20 8 4 667 14 5 164 29 12 4 1000 16 8 176 9 9 3 3330 19 3 187 25 10 3 5000 16 5 188 7 8 0 Positive Control*** 193 73 719 54 321 24

NC = No count due to unacceptable mean vehicle control value. See retest data experiment ID 15437-B2 ** TA98 2-aminoanthracene 2.5 g/plate *** TA98 2-nitrofluorene 1.0 g/plate

TA100 2-aminoanthracene 2.5 g/plate TA100 sodium azide 2.0 g/plate TA1535 2-aminoanthracene 2.5 g/plate TA1535 sodium azide 2.0 g/plate TA1537 2-aminoanthracene 2.5 g/plate TA1537 ICR-191 2.0 g/plate TA1538 2-aminoanthracene 2.5 g/plate TA1538 2-nitrofluorene 1.0 g/plate




Assay 1: Experiment ID 15437-B1 (Continued)

MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION Metabolic Activation


MEAN S.D. MEAN S.D. Microsomes: rat liver Vehicle Control 8 1 20 7 Test Article 100 9 2 20 3 333 8 3 20 7 667 6 3 22 10 1000 6 1 20 3 3330 5 2 24 3 5000 6 1 19 7 Positive Control** 128 7 1189 58 Microsome: none Vehicle Control 5 4 14 3 Test Article 100 3 1 9 1 333 9 2 12 4 667 5 2 15 3 1000 7 4 13 1 3330 5 1 10 2 5000 7 2 10 1 Positive Control*** 267 32 327 52

** TA98 2-aminoanthracene 2.5 g/plate *** TA98 2-nitrofluorene 1.0 g/plate TA100 2-aminoanthracene 2.5 g/plate TA100 sodium azide 2.0 g/plate TA1535 2-aminoanthracene 2.5 g/plate TA1535 sodium azide 2.0 g/plate TA1537 2-aminoanthracene 2.5 g/plate TA1537 ICR-191 2.0 g/plate TA1538 2-aminoanthracene 2.5 g/plate TA1538 2-nitrofluorene 1.0 g/plate




Assay 2: Experiment ID 15437-B2 REVERTANTS PER PLATE Metabolic Activation


1 2 3 MEAN S.D. Microsomes: rat liver Vehicle control 104 116 94 105 11 Test Article 100 125 95 107 109 15 333 115 110 94 106 11 667 129 110 100 113 15 1000 138 113 NC 126 18 3330 131 125 121 126 5 5000 156 123 97 125 30 Positive Control** 1092 1289 1468 1283 188

NC = No count due to contamination on the plate ** TA100 2-aminoanthracene 2.5 g/plate

2.6.7.8.C. Genotoxicity: In Vitro Report Title: Mutagenicity Test of FTC Using Microorganisms


AF2: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide ICR-191: 2-Methoxy-6-chloro-9[3-(2-chloroethyl)-aminopropylamino]acridine-2HCl 2AA: 2-Aminoanthracene CONFIDENTIAL Page 165 17AUG2010

2.6.7.8.C. K01-3154: Mutagenicity Test of FTC Using Microorganisms

Test for Induction of: Reverse mutation in bacterial cells

No. of Independent Assays: 2 Study No.: K01-3154

Species/Strain: S. typhimurium (TA98, TA100, TA1535, TA1537), E. coli(WP2 uvr A)

No. of Replicate Cultures: 3 (all negative controls), 2 (#1), 2 (#2)

Metabolizing System:

Phenobarbital and 5, 6-benzoflavone induced rat liver S9

GLP Compliance: Yes

Vehicles: For Test Article: Distilled water For Positive Controls: DMSO, except distilled water for sodium azide Treatments: Pre-incubation method for initial (#1) and confirmatory (#2) assays Date of Treatments: 20 ,

20 Cytotoxic Effects: No appreciable toxicity was observed. Genotoxic Effects: None

Metabolic Test Article Dose Level Assay No. 1 Initial Mutagenicity Assay

Revertant Colony Counts (Mean) Activation ( g/plate) TA100 TA1535 WP2 uvr A TA98 TA1537 Without (Vehicle) -- 131 13 30 26 13 Activation Test Article 4.88 137 10 33 21 9 19.5 127 9 35 24 14 78.1 129 14 26 20 14 313 118 8 32 23 15 1250 123 10 35 23 11 5000 125 12 33 19 13 AF-2 0.01 523 284 Sodium azide 0.5 451 AF-2 0.1 509 ICR-191 0.5 1139




Metabolic Test Article Dose Level Assay No. 1 Initial Mutagenicity Assay

Revertant Colony Counts (Mean) Activation ( g/plate) TA100 TA1535 WP2 uvr A TA98 TA1537 With (Vehicle) -- 149 8 34 27 31 Activation Test Article 4.88 148 7 34 40 21 19.5 140 11 40 37 23 78.1 135 6 37 31 26 313 135 9 32 29 23 1250 128 5 41 37 29 5000 137 10 40 33 22 2AA 1 772 2AA 2 203 213 2AA 10 565 2AA 0.5 312

Metabolic Test Article Dose Level Assay No. 2 Confirmatory Mutagenicity Assay

Revertant Colony Counts (Mean) Activation ( g/plate) TA100 TA1535 WP2 uvr A TA98 TA1537 Without (Vehicle) -- 117 10 20 19 8 Activation Test Article 313 110 8 23 20 12 625 118 9 26 25 10 1250 112 12 15 17 12 2500 115 7 22 16 12 5000 111 9 26 18 10 AF-2 0.01 471 216 Sodium azide 0.5 411 AF-2 0.1 484 ICR-191 0.5 1270




Metabolic Test Article Dose Level Assay No. 2 Confirmatory Mutagenicity Assay

Revertant Colony Counts (Mean) Activation ( g/plate) TA100 TA1535 WP2 uvr A TA98 TA1537 With (Vehicle) -- 121 8 23 25 19 Activation Test Article 313 132 9 24 32 20 625 118 7 21 35 28 1250 137 10 26 25 22 2500 123 14 29 33 14 5000 127 9 27 28 16 2AA 1 650 2AA 2 178 172 2AA 10 538 2AA 0.5 381

2.6.7.8.D. Genotoxicity: In Vitro Report Title: In Vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay)



2.6.7.8.D. TOX012: In Vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay)

Test for Induction of: Forward mutations at the thymidine kinase locus in cultured L5178Y cells. Mutants are unable to metabolise the non-toxic thymidine metabolite, trifluorothymidine (TFT) to its lethal metabolite and hence are resistant to its lethal effect.

No. of Independent Assays: Preliminary Toxicity assay: 1 experiment Mutagenicity assay: 1 experiment

Study No: TOX012

Strains: L5178Y mouse lymphoma cells TK+/- No. of Replicate Cultures: A Preliminary Toxicity assay a) single b) single Mutagenicity assay a) duplicate b) duplicate

Metabolizing System: Aroclor 1254-induced rat liver (S9 mix)

No. of Cells Analyzed/Culture: Adjusted to 3 x 105/mL at 24-48 hours after treatment Cultures less than 3 x 105/mL were not adjusted

Vehicles: Sterile distilled water For Test Article: FTC (lot + (± S9))

For Positive Controls: a) - S9: Methyl methanesulfonate (MMS) 10 and 20 g/mLb) + S9: 7,12-Dimethyl-benz(a)anthracene (7,12-DMBA) 2.5 and 4.0 g/mL

GLP Compliance: Yes

Treatment: Treatment period was for 4 hours at 37 1°C. Recovery at approximately 24 and 48 hours after treatment ( S9).

Date of Treatment: , 19 – , 19

Cytotoxic Effects: Suspension growth relative to the solvent controls at 5000 g/mL was 76% without activation and 99% with S9 activation. Genotoxic Effects: Emtricitabine was concluded to be negative in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.

A Preliminary Toxicity assay: a) - S9: 0.5, 1.5, 5.0, 15, 50, 150, 500, 1500, 5000 g/mL. Vehicle control. b) + S9: (same) Mutagenicity assay: a) - S9: 1000, 2000, 3000, 4000, 5000 g/mL. Positive and vehicle controls. b) +S9: (same)




Assay 1: Study No. G97BG07.702

MUTATION ASSAY WITHOUT ACTIVATION

Test Condition Daily Cell Counts

(conc.106cells)

Mutant Colonies

Avg.

Suspension Growtha ATotalb

Suspension Growth Controlc

(%)

Viable Colonies

(VC) Avg.

Cloning Growth Avg. VC

Cloning Growth Controld

(%)

Total Growthe

(%)

Mutant Frequencyf

(106 cells) Day 1 Day 2 Vehicle Control 1 1.475 1.514 48 24.8 173 173 - 55 Vehicle Control 2 1.398 1.502 51 23.3 172 172 - 60 Positive Control 1

Methyl Methanesulfonate ( g/mL) 10 1.029 1.356 224 15.5 64 105 105 61 39 426 20 0.885 0.910 218 8.9 37 50 50 29 11 865

FTC ( g/mL) 1000 A 1.245 1.575 45 21.8 91 174 174 101 91 52

B 1.304 1.631 39 23.6 98 141 141 82 80 55 2000 A 1.180 1.611 55 21.1 88 165 165 96 84 66

B 1.221 1.467 58 19.9 83 146 146 85 70 79 3000 A 1.098 1.641 53 20.0 83 176 176 102 85 61

B 1.081 1.557 54 18.7 78 145 145 84 65 74 4000 A 1.064 1.502 55 17.7 74 143 143 83 61 77

B 0.945 1.505 59 15.8 66 142 142 82 54 83 5000 A 0.856 1.410 54 13.4 56 166 166 96 54 66

B 0.874 1.387 44 13.5 56 161 161 93 52 55 A and B or 1 and 2 are duplicate cultures a Cultures containing 0.3 106 cells/mL on day 1 and 2 are considered as having 0% total suspension growth. b Total suspension growth = (Day 1 cell conc./0.3 106 cells/mL) (Day 2 cell conc./Day 1 adjusted cell conc.) c % of control growth = (total treatment suspension growth / average solvent control total suspension growth) 100 d % control cloning growth = (average V.C. of treated culture / average V.C. of solvent control) 100 e % total growth = (% suspension growth % cloning growth) / 100 f Mutant frequency (per 106 surviving cells) = (Average # TFT colonies / average # V.C. colonies) 200




Assay 1: Study No. G97BG07.702 MUTATION ASSAY WITH ACTIVATION

Test Condition Daily Cell Counts (conc.106cells)

Mutant Colonies

Avg.

Suspension Growtha

Totalb

Suspension Growth Controlc

(%)

Viable Colonies

(VC) Avg.

Cloning Growth Avg. VC

Cloning Growth Controld

(%)

Total Growthe

(%)

Mutant Frequencyf (106 cells)

Day 1 Day 2 Vehicle Control 1 1.124 1.510 51 18.9 130 130 79 Vehicle Control 2 1.052 1.517 40 17.7 171 171 47 Positive Control 1

7,12-Dimethyl-benz(a)anthracene ( g/mL) 2.5 0.816 1.467 158 13.3 73 112 112 74 54 282

4 0.640 0.993 231 7.1 39 87 87 57 22 534 FTC ( g/mL)

1000 A 1.178 1.530 42 20.0 109 159 159 106 116 53 B 1.163 1.525 42 19.7 108 144 144 96 103 58

2000 A 1.038 1.568 33 18.1 99 132 132 88 87 50 B 1.100 1.513 43 18.5 101 117 117 78 78 74

3000 A 1.106 1.475 50 18.1 99 166 166 110 109 61 B 1.103 1.390 53 17.0 93 138 138 91 85 77

4000 A 1.055 1.450 46 17.0 93 153 153 101 94 60 B 1.058 1.529 46 18.0 98 170 170 112 111 54

5000 A 1.072 1.338 52 15.9 87 171 171 113 99 61 B 1.001 1.571 52 17.5 96 129 129 86 82 81

A and B or 1 and 2 are duplicate cultures a Cultures containing 0.3 106 cells/mL on day 1 and 2 are considered as having 0% total suspension growth. b Total suspension growth = (Day 1 cell conc./0.3 106 cells/mL) (Day 2 cell conc./Day 1 adjusted cell conc.) c % of control growth = (total treatment suspension growth / average solvent control total suspension growth) 100 d % control cloning growth = (average V.C. of treated culture/average V.C. of solvent control) 100 e % total growth = (% suspension growth % cloning growth)/100 f Mutant frequency (per 106 surviving cells) = (Average # TFT colonies / average # V.C. colonies) 200

2.6.7.8.E. Genotoxicity: In Vitro Report Title: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium



2.6.7.8.E. TMC278-Exp5540: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium

Test: In vitro Ames test in Salmonella typhimurium Species/strain: Salmonella typhimurium strain TA1535, TA1537, TA98, TA100, TA102 Metabolizing system: Induced rat (Aroclor 1254) liver metabolic system S9-mix No. of independent assays: 2 No. of replicate cultures: 3

Treatment: 48 hours Concentrations: 0, 7.81, 15.63, 31.25, 62.50, 125, 250 and 500 μg/plate TMC278 base Test article batch: Vehicle test article: DMSO Positive controls: 2-nitrofluorene, sodium azide, 9-aminoacridine, 2-aminoanthracene, 4-nitroquinoline-N-oxide

Testing facility: J&J PRD Study No.: TMC278-Exp5540 GLP compliance: Yes

Cytotoxic effects: no - strong precipitation limited the dose Genotoxic effects: no genotoxic effects


Control, Test and Reference Articles

Concentration (μg/plate)

Assay # 1 Revertant Colony Counts (Mean SD)

TA98 TA1537 TA100 TA1535 TA102 DMSO 0.00 16 ± 2.1 4 ± 1.2 112 ± 25.9 11 ± 2.1 229 ± 20.0

7.81 17 ± 2.0 9 ± 2.3 96 ± 3.8 12 ± 4.4 212 ± 18.8 15.63 12 ± 2.1 8 ± 1.2 97 ± 11.5 14 ± 1.7 221 ± 16.5 31.25 21 ± 1.4 6 ± 1.2 a 93 ± 8.1 a 10 ± 3.5 a 208 ± 31.2 62.50 13 ± 4.0a 5 ± 2.3 a 79 ± 9.6 a 14 ± 2.5 a 187 ± 17.0 a 125.00 11 ± 5.3a 6 ± 2.0 a 88 ± 10.1 a 11 ± 4.4 a 179 ± 13.5 a 250.00 15 ± 4.6a 4 ± 1.0 a 94 ± 8.5 a 12 ± 4.0 a 173 ± 31.4 a

TMC278

500.00 17 ± 1.7a 6 ± 2.9 a 77 ± 7.5 a 8 ± 1.5 a 173 ± 10.1 a 2- Nitrofluorene 5.00 640 ± 68.4 Sodium Azide 1.00 699 ± 28.1 344 ± 76.3

9-Amino-Acridine 50.00 365 ± 153.1

Without S9

4-Nitroquinoline-N-Oxide 5.00 1224 ± 251.8 a Precipitation at this dose level and above; DMSO: dimethyl sulfoxide.




Study: TMC278-Exp5540: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium (continued)





TA98 TA1537 TA100 TA1535 TA102 DMSO 0.00 17 ± 3.2 9 ± 4.4 94 ± 4.4 12 ± 1.2 344 ± 42.1

7.81 26 ± 5.1 5 ± 4.0 90 ± 3.0 12 ± 1.5 293 ± 9.2 15.63 27 ± 5.3 7 ± 3.5 115 ± 12.3 11 ± 0.6 288 ± 34.9 31.25 20 ± 5.7 a 11 ± 0.6 a 97 ± 12.1 a 12 ± 1.5 a 393 ± 49.7 a 62.50 17 ± 3.6 a 7 ± 4.4 a 84 ± 10.4 a 10 ± 4.4 a 356 ± 10.6 a 125.00 17 ± 10.6 a 8 ± 4.0 a 89 ± 12.0 a 7 ± 4.0 a 251 ± 58.0 a 250.00 22 ± 1.2 a 5 ± 1.5 a 87 ± 21.5 a 10 ± 0.6 a 248 ± 18.3 a

TMC278

500.00 18 ± 4.0 a 4 ± 1.2 a 67 ± 7.0 a 9 ± 6.0 a 207 ± 35.9 a 1.00 837 ± 122.2 - 784 ± 71.1 144 ± 20.0 -

With S9 (20 L)

2-Amino-Anthracene 2.50 - 276 ± 34.9 - - 1000 ± 194.0

a precipitation at this dose level and above; DMSO: dimethyl sulfoxide.




Study: TMC278-Exp5540: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium (continued) Metabolic Activation




TA98 TA1537 TA100 TA1535 TA102 DMSO 0.00 18 ± 2.6 9 ± 0.6 95 ± 12.5 11 ± 2.0 248 ± 31.2

7.81 16 ± 9.3 8 ± 2.6 86 ± 11.1 9 ± 2.3 201 ± 16.2 15.63 16 ± 6.6 9 ± 1.0 76 ± 16.8 10 ± 2.1 260 ± 69.4 31.25 17 ± 5.1 a 9 ± 5.1 94 ± 15.6 13 ± 5.5 252 ± 14.4 a 62.50 15 ± 3.6 a 9 ± 5.5 a 93 ± 8.4 a 14 ± 3.5 a 315 ± 36.1 a 125.00 15 ± 1.2 a 7 ± 2.1 a 96 ± 10.2 a 8 ± 1.0 a 284 ± 39.4 a 250.00 15 ± 4.2 a 8 ± 2.6 a 104 ± 12.0 a 13 ± 4.7 a 171 ± 14.0 a

TMC278

500.00 16 ± 1.5 a 8 ± 1.5 a 105 ± 15.9 a 11 ± 2.0 a 172 ± 20.0 a 2- Nitrofluorene 5.00 661 ± 105.6 - - - - Sodium Azide 1.00 - - 837 ± 82.1 253 ± 41.1 -

9-Amino-Acridine 50.00 - 144 ± 20.8 - - -

Without S9

4-Nitroquinoline-N-Oxide 5.00 - - - 1071 ± 208.7 DMSO 0.00 19 ± 2.9 7 ± 1.2 95 ± 13.6 14 ± 3.1 337 ± 34.9

7.81 33 ± 6.2 5 ± 1.0 115 ± 11.5 9 ± 1.7 353 ± 26.0 15.63 33 ± 4.6 7 ± 0.6 90 ± 20.2 8 ± 3.1 385 ± 26.6 31.25 31 ± 1.2 a 7 ± 2.5 108 ± 12.1 10 ± 2.1 393 ± 34.9 a 62.50 29 ± 3.6 a 10 ± 3.5 a 92 ± 5.6 a 8 ± 1.7 a 295 ± 58.3 a 125.00 34 ± 4.7 a 6 ± 3.5 a 94 ± 4.2 a 11 ± 3.1 a 261 ± 38.9 a 250.00 29 ± 9.6 a 6 ± 0.6 a 88 ± 0.6 a 5 ± 4.0 a 180 ± 4.0 a

TMC278

500.00 33 ± 2.5 a 5 ± 2.0 a 65 ± 11.0 a 8 ± 3.5 a 124 ± 21.2 a 1.00 215 ± 112.0 - 435 ± 20.1 64 ± 5.5 -

With S9 (50 L)

2-Amino-Anthracene 2.50 - 97 ± 6.4 - - 1184 ± 284.1

a precipitation at this dose level and above; DMSO: dimethyl sulfoxide.

2.6.7.8.F. Genotoxicity: In Vitro Report Title: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium



2.6.7.8.F. TMC278-Exp5693: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium

Test: In Vitro Mammalian Forward Mutation Test Species/strain: Salmonella typhimurium, strains TA1535, TA1537, TA98, TA100, TA102 Metabolizing System: Induced rat (Aroclor 1254) liver metabolic system S9-mix No of independent assays: 2 No of replicate cultures: 3

Treatment: 48 hours Concentrations: 7.81, 15.63, 31.25, 62.50, 125, 250 and 500 μg/plate TMC278 base Test article batch: Vehicle test article: DMSO Positive controls: 2-nitrofluorene, sodium azide, 9-aminoacridine, 2-aminoanthracene, 4-nitroquinoline-N-oxide


Cytotoxic effects: Weak decrease in revertants with some strains Genotoxic effects: No genotoxic effects

Assay # 1

Revertant Colony Counts (Mean ± SD) Metabolic Activation



TA98 TA1537 TA100 TA1535 TA102 DMSO 0.00 12 ± 1.0 7 ± 3.2 108 ± 8.5 9 ± 0.6 291 ± 6.1

7.81 10 ± 3.8 6 ± 3.5 131 ± 22.6 13 ± 2.5 285 ± 12.2 15.63 13 ± 4.0 10 ± 0.6 146 ± 39.1 11 ± 2.3 311 ± 41.6 31.25 11 ± 2.6 8 ± 3.8 127 ± 18.2a 11 ± 0.6 313 ± 45.5 62.50 9 ± 0.6a 13 ± 1.2a 124 ± 13.2a 10 ± 3.6a 299 ± 43.1a

125.00 11 ± 3.2a 8 ± 1.2a 128 ± 22.3a 11 ± 1.0a 257 ± 36.3a 250.00 10 ± 4.0a 8 ± 2.3a 105 ± 25.7a 14 ± 3.2a 275 ± 8.3a

TMC278

500.00 12 ± 0.6a 12 ± 4.7a 99 ± 12.5a 8 ± 3.6a 261 ± 12.2a 2-Nitrofluorene 5.00 840 ± 254.0 - - - - Sodium Azide 1.00 - - 469 ± 60.6 207 ± 37.2 -

9-Amino-Acridine 50.00 123 ± 35.9 - - -

Without S9

4-Nitroquinoline-N-Oxide 5.00 - - 2581 ± 531.9 a Precipitation at this dose level and above; DMSO: dimethyl sulfoxide




Study: TMC278-Exp5693: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium (continued)

Assay # 1 Revertant Colony Counts (Mean ± SD) Metabolic

Activation Control, Test and Reference Articles


TA98 TA1537 TA100 TA1535 TA102 DMSO 0.00 23 ± 4.6 9 ± 1.2 118 ± 19.5 9 ± 2.5 365 ± 20.1

7.81 23 ± 7.4 6 ± 2.1 124 ± 1.5 9 ± 1.5 327 ± 36.3 15.63 25 ± 9.5 10 ± 4.6 127 ± 12.7 12 ± 7.4 249 ± 44.1 31.25 19 ± 1.5 10 ± 0.6a 121 ± 6.4a 11 243 ± 42.4 62.50 25 ± 4.6a 9 ± 3.5a 91 ± 8.7a 8 ± 5.5a 283 ± 10.1a

125.00 20 ± 4.0a 8 ± 4.0a 80 ± 14.5 a 9 ± 3.2a 412 ± 91.7a 250.00 17 ± 10.7a 6 ± 0.6a 117 ± 6.5a 7 ± 2.0a 259 ± 83.9a

TMC278

500.00 9 ± 1.7a 8 ± 2.1a 109 ± 10.4a 11 ± 3.0a 147 ± 44.1a 1.00 1161 ± 160.5 1765 ± 180.0 211 ± 26.6

With S9 (20 L)

2-Amino-anthracene 2.50 - 344 ± 72.0 - - 1279 ± 47.4

a Precipitation at this dose level and above; DMSO: dimethyl sulfoxide




Study: TMC278-Exp5693: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium (continued) Assay # 2

Revertant Colony Counts (Mean ± SD) Metabolic Activation



TA98 TA1537 TA100 TA1535 TA102 DMSO 0.00 13 ± 2.0 8 ± 2.6 121 ± 14.5 12 ± 4.0 228 ± 42.1

7.81 15 ± 3.5 8 ± 2.5 129 ± 9.2 15 ± 1.5 292 ± 72.8 15.63 17 ± 2.5 10 ± 2.9 145 ± 24.8 17 ± 2.6 284 ± 52.0 31.25 13 ± 5.5a 7 ± 1.0a 109 ± 5.0 a 15 ± 4.0a 261 ± 36.3 62.50 14 ± 4.0a 6 ± 2.1a 118 ± 11.1a 13 ± 4.9 a 255 ± 33.3a

125.00 16 ± 4.6a 7 ± 2.5a 145 ± 21.0a 15 ± 4.7a 279 ± 12.9a 250.00 11 ± 4.6a 6 ± 2.5a 123 ± 5.6a 15 ± 2.1a 200 ± 6.9a

TMC278

500.00 14 ± 3.5a 8 ± 4.4a 113 ± 6.5a 14 ± 2.5a 225 ± 18.0a 2-Nitrofluorene 5.00 805 ± 48.9 - - - - Sodium Azide 1.00 - - 473 ± 16.2 472 ± 28.8 -

9-Amino-Acridine 50.00 - 84 ± 4.4 - - -

Without S9

4-Nitroquinoline-N-Oxide 5.00 - - - - 2363 ± 352.8 DMSO 0.00 20 ± 6.4 8 ± 2.1 121 ± 12.3 15 ± 2.5 340 ± 14.4

7.81 21 ± 7.0 9 ± 2.9 128 ± 11.2 10 ± 2.1 343 ± 76.4 15.63 24 ± 2.0 9 ± 5.5 140 ± 12.3 12 ± 2.1 377 ± 63.8 31.25 27 ± 6.0 9 ± 1.7 115 ± 7.5 10 ± 2.9 309 ± 32.3 62.50 27 ± 5.0a 11 ± 5.0 131 ± 6.0a 9 ± 3.1a 275 ± 30.6

125.00 18 ± 2.6a 12 ± 1.5a 115 ± 5.7a 8 ± 4.2a 328 ± 59.2a 250.00 21 ± 6.0a 8 ± 1.5a 130 ± 4.2a 12 ± 3.5a 304 ± 52.5a

TMC278

500.00 19 ± 7.2a 10 ± 3.1a 126 ± 4.5a 8 ± 3.5a 263 ± 22.0a 1.00 320 ± 32.7 - 537 ± 54.3 103 ± 14.7 -

With S9 (50 L)

2-Amino-anthracene 2.50 - 94 ± 4.6 - - 1475 ± 20.1

a Precipitation at this dose level and above; DMSO: dimethyl sulfoxide

2.6.7.8.G. Genotoxicity: In Vitro Report Title: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium



2.6.7.8.G. TMC278-NC335: In Vitro Bacterial Reverse Mutation Test with R314585 (TMC278.HCl) in Salmonella typhimurium

Test: In Vitro Bacterial Reverse Mutation Test Species/strain: S. Typhimurium strains TA98, TA1537, TA100, TA1535 and TA102 Metabolizing System: Induced rat (Aroclor 1254) liver metabolic system S9-mix No of independent assays: 2 No of replicate cultures: 3 No of cells analyzed/culture: 10

Treatment: 48 to 72hours Concentrations: 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 g/plate TMC278.HCl Test article batch: Vehicle test article: DMSO Vehicle positive controls: DMSO and water Positive control: 2-nitrofluorene, sodium azide, 9-aminoacridine, 2-aminoanthracene, 4-nitroquinoline-N-oxide

Testing facility: J&J PRD Study No.: TMC278-NC335 GLP compliance: Yes

Cytotoxic effects: No Genotoxic effects: No

Assay # 1 (Revertant Colony Counts (Mean ± SD)

Metabolic Activation Test Article Concentration

(μg/plate)a TA98 TA100 TA1535 TA1537 TA102

DMSO 0 15.7 ± 2.1 119.0 ± 18.0 11.3 ± 2.1 11.0 ± 2.6 365.3 ± 10.7 3.91 11.7 ± 1.5 101.3 ± 7.6 9.0 ± 1.0 11.7 ± 3.5 382.7 ± 5.0 7.81 11.3 ± 3.5 109.0 ± 16.4 6.7 ± 4.7 9.7 ± 2.1 378.0 ± 9.8

15.63 22.7 ± 3.8 129.0 ± 4.6 8.0 ± 2.6 8.0 ± 3.0 363.3 ± 26.3 31.25 20.7 ± 3.2 121.7 ± 23.7 9.7 ± 3.5 6.0 ± 2.6 378.7 ± 27.9 62.5 13.7 ± 5.9 127.0 ± 16.7 7.3 ± 0.6 5.3 ± 1.2 357.3 ± 18.9 125 15.7 ± 4.7 P1 110.0 ± 11.8 P1 8.3 ± 2.1 P1 6.0 ± 2.6 P1 364.0 ± 20.3 P1

TMC278

250 19.7 ± 4.0 P1 106.3 ± 14.4 P1 9.7 ± 3.2 P1 5.7 ± 1.2 P1 303.0 ± 18.2 P1 2-Nitrofluorene 5.00 514.7 ± 146.8 * - - - - Sodium Azide 1.00 - 536.0 ± 4.4 * 478.3 ± 44.5 * - -

9-Amino-Acridine 50.00 - - - 887.0 ± 177.2 * -

Without S9

4-Nitroquinoline-N-Oxide 5.00 - - - - 3309.3 ± 44.8 *

* more than 2-fold increase with TA98, TA100 and TA102 compared to the vehicle control, more than 3-fold increase with TA1535 and TA1537 compared to the vehicle control, P1: Slight precipitation, DMSO: dimethyl sulfoxide, -: not applicable




TMC278-NC335: In Vitro Bacterial Reverse Mutation Test with R314585 (TMC278.HCl) in Salmonella typhimurium (continued) Assay # 1 (Revertant Colony Counts (Mean ± SD)) Metabolic Activation

Test Article

Concentration (μg/plate)a TA98 TA100 TA1535 TA1537 TA102

DMSO 0 16.3 ± 2.5 150.7 ± 19.0 14.3 ± 1.2 4.0 ± 0.0 415.3 ± 51.0 3.91 15.7 ± 2.5 163.3 ± 31.5 11.3 ± 0.6 4.0 ± 1.7 476.0 ± 7.2 7.81 21.7 ± 9.6 176.7 ± 13.9 12.3 ± 4.7 5.5 ± 3.5 448.0 ± 37.5

15.63 19.7 ± 4.7 161.3 ± 2.5 13.0 ± 4.4 6.0 ± 2.0 456.3 ± 55.6 31.25 19.3 ± 5.0 180.0 ± 7.8 7.7 ± 0.6 4.7 ± 2.5 478.0 ± 32.1 62.5 21.0 ± 3.6 222.3 ± 15.2 11.7 ± 0.6 5.3 ± 1.2 483.0 ± 30.3 125 20.3 ± 3.8 P1 171.3 ± 15.5 P1 10.0 ± 4.6 P1 6.0 ± 4.2 P1 387.0 ± 67.7 P1

TMC278

250 22.0 ± 8.5 P2 163.0 ± 14.5 P2 8.7 ± 4.0 P2 4.3 ± 0.6 P2 238.7 ± 71.1 P2 2.5 1244.0 ± 79.5 * 1102.3 ± 338.5 * 116.7 ± 5.5 * 124.0 ± 16.5 * -

With S9

2-Amino-anthracene 7.5 - - - - 2516.7 ± 41.0 *

a unless stated otherwise, *more than 2-fold increase with TA98, TA100 and TA102 compared to the vehicle control, more than 3-fold increase with TA1535 and TA1537 compared to the vehicle control, P1: Slight precipitation, P2: Moderate precipitation, DMSO: dimethyl sulfoxide, -: not applicable




TMC278-NC335: In Vitro Bacterial Reverse Mutation Test with R314585 (TMC278.HCl) in Salmonella typhimurium (continued) Assay # 2 (Revertant Colony Counts (Mean ± SD) Metabolic Activation Test Article Concentration

(μg/plate)a TA98 TA100 TA1535 TA1537 TA102

DMSO 0 19.3 ± 3.8 113.7 ± 3.2 7.0 ± 2.6 7.3 ± 0.6 288.0 ± 19.1 3.91 14.0 ± 4.6 97.3 ± 10.1 7.3 ± 2.5 4.7 ± 1.5 303.3 ± 7.6 7.81 13.0 ± 7.8 96.3 ± 21.5 7.3 ± 4.0 8.0 ± 4.4 339.7 ± 12.7

15.63 14.7 ± 2.5 106.0 ± 18.4 5.3 ± 2.3 7.3 ± 2.5 341.0 ± 21.6 31.25 14.7 ± 6.5 114.0 ± 6.1 7.3 ± 3.1 6.7 ± 3.8 295.3 ± 13.4 62.5 15.0 ± 5.0 97.3 ± 8.4 7.3 ± 4.2 8.3 ± 1.2 292.7 ± 22.5 125 19.0 ± 7.0 113.7 ± 3.8 5.0 ± 2.6 5.3 ± 3.8 288.3 ± 20.5

TMC278

250 18.0 ± 3.6 104.3 ± 7.8 6.3 ± 3.5 7.7 ± 3.5 278.7 ± 13.3 2-Nitrofluorene 5.00 1048.7 ± 44.8 * - - - - Sodium Azide 1.00 - 415.7 ± 2.1 * 313.0 ± 10.4 * - -

9-Amino-Acridine 50.00 - - - 124.3 ± 39.5 * -

Without S9

4-Nitroquinoline-N-Oxide 5.00 - - - - 3250.0 ± 168.1 *

a unless stated otherwise; *more than 2-fold increase with TA98, TA100 and TA102 compared to the vehicle control, more than 3-fold increase with TA1535 and TA1537 compared to the vehicle control; DMSO: dimethyl sulfoxide; -: not applicable




TMC278-NC335: In Vitro Bacterial Reverse Mutation Test with R314585 (TMC278.HCl) in Salmonella typhimurium (continued) Assay # 2 (Revertant Colony Counts (Mean ± SD) Metabolic Activation

Test Article

Concentration (μg/plate)a TA98 TA100 TA1535 TA1537 TA102

DMSO 0 19.3 ± 3.5 135.0 ± 11.3 9.0 ± 1.0 6.7 ± 1.5 363.7 ± 42.7 3.91 29.7 ± 3.5 125.0 ± 13.5 9.0 ± 5.2 4.7 ± 3.8 385.3 ± 29.3 7.81 25.0 ± 5.3 129.3 ± 4.7 7.3 ± 0.6 2.7 ± 1.5 389.3 ± 25.1

15.63 25.0 ± 4.4 182.3 ± 26.5 8.7 ± 5.7 5.7 ± 1.5 406.0 ± 19.1 31.25 26.7 ± 1.2 147.3 ± 14.6 7.7 ± 1.2 3.7 ± 1.2 417.7 ± 9.9 62.5 30.3 ± 7.2 P1 139.7 ± 15.1 P1 9.7 ± 1.2 P1 8.3 ± 4.9 358.3 ± 17.2 125 23.0 ± 4.6 P1 152.0 ± 4.6 P1 9.7 ± 1.2 P1 9.7 ± 1.5 P1 268.0 ± 18.3 P1

TMC278

250 28.7 ± 5.5 P2 126.0 ± 4.6 P2 7.3 ± 0.6 P2 5.3 ± 1.5 P2 205.3 ± 32.1 P2

With S9

2-Amino-anthracene 7.5 2417.3 ± 156.2 * 2217.7 ± 28.5 * 130.0 ± 7.0 * 242.3 ± 9.5 * 1228.7 ± 90.1 *

a unless stated otherwise; *more than 2-fold increase with TA98, TA100 and TA102 compared to the vehicle control, more than 3-fold increase with TA1535 and TA1537 compared to the vehicle control; P1: Slight precipitation; P2: Moderate precipitation; DMSO: dimethyl sulfoxide

2.6.7.8.H. Genotoxicity: In Vitro Report Title: In Vitro Bacterial Reverse Mutation Test with Salmonella typhimurium with human liver S9-mix



2.6.7.8.H. TMC278-NC279: In Vitro Bacterial Reverse Mutation Test of TMC278 in Salmonella typhimurium with human liver S9-mix

Test: In Vitro Bacterial Reverse Mutation Test Species/strain: Salmonella Typhimurium strains TA98, TA1537, TA100, TA1535 and TA102 Metabolizing System: Human S9_Mix No of independent assays: 4 No of replicate cultures: 3

Treatment: 48 to 72h Concentrations: of 0, 3, 7.81, 10, 15.63, 25, 31.25, 50, 62.5, 100, 125, 200, 250, 400 and 500

g/plate TMC278 base Test article batch: Vehicle test article: DMSO Vehicle positive controls: DMSO and water

Testing facility: J&J PRD Study no.: TMC278-NC279 GLP compliance: Yes

Positive controls: 2-nitrofluorene, sodium azide, 9-aminoacridine, 2-aminoanthracene, 4-nitroquinoline-N-oxide

Cytotoxic effects: Weak Genotoxic effects: No

Assay # 1 (Revertant Colony Counts (Mean ± SD) Metabolic Activation Test Article Concentration

(μg/plate)a TA98 TA100a TA1535 TA1537

DMSO 0 14.3 ± 1.2 99.0 ± 7.5 11.7 ± 5.0 5.7 ± 3.2 2-Nitrofluorene 5.00 557.7 ± 64.8* - - -

1.00 - - 411.0 ± 26.5* - Sodium Azide 2.50 - 796.7 ± 26.5* - -

9-Aminoacridine 50.00 - - - 20.3 ± 2.1*

Without S9

4-Nitroquinoline-N-Oxide 5.00 - - - - a pre-incubation; * more than 2-fold increase with TA98 and TA100 compared to the vehicle control, more than 3-fold with TA1535 and TA1537 compared to the vehicle

control; DMSO: dimethyl sulfoxide; -: Not Applicable




Study TMC278-NC279: In Vitro Bacterial Reverse Mutation Test of TMC278 in Salmonella typhimurium with human liver S9-mix (continued) Assay # 1 (Revertant Colony Counts (Mean ± SD))

Metabolic Activation Test Article Concentration (μg/plate)a TA98 TA100 TA1535 TA1537

DMSO 0 25.7 ± 7.8 156.3 ± 17.6 11.0 ± 4.4 13.7 ± 3.8 7.81 33.3 ± 5.7 135.7 ± 17.2 11.3 ± 3.1 16.7 ± 2.1

15.63 28.0 ± 7.5 153.0 ± 6.9 10.0 ± 4.0 13.7 ± 5.5 31.25 31.3 ± 5.9 145.3 ± 4.7 17.7 ± 8.5 19.7 ± 3.8 62.5 19.0 ± 10.8 158.7 ± 28.1 16.0 ± 3.6 30.3 ± 4.9 125 18.0 ± 1.7 P 161.3 ± 25.6 15.7 ± 7.5 P 26.7 ± 10.2 P 250 17.0 ± 7.0 P 163.3 ± 27.2 P 10.3 ± 3.1 P 33.0 ± 11.3 P

With S9 TMC278

500 15.7 ± 1.2 P 132.0 ± 6.0 P 9.3 ± 4.6 P 25.7 ± 10.0 P a pre-incubation; * more than 2-fold increase with TA98 and TA100 compared to the vehicle control, more than 3-fold with TA1535 and TA1537 compared to the vehicle

control; DMSO: dimethyl sulfoxide; -: Not Applicable; P: Precipitation.






DMSO 0 20.3 ± 0.6 141.0 ± 3.5 20.3 ± 4.9 367.7 ± 18.3 2-Nitrofluorene 5.00 386.0 ± 20.7* - - -

1.00 - - 610.0 ± 27.6* - Sodium Azide 2.50 - 1115.3 ± 40.4* - -

Without S9

4-Nitroquinoline-N-Oxide

5.00 - - - 2127.3 ± 122.2*

a unless stated otherwise; * more than 2-fold increase with TA98, TA100 and TA102 and more than 3-fold with TA1535 compared to the vehicle control; DMSO: dimethyl sulfoxide; -: Not Applicable






DMSO 0 34.7 ± 8.0 143.3 ± 16.3 24.0 ± 6.6 479.3 ± 44.2 3 31.0 ± 5.0 150.0 ± 9.6 22.7 ± 3.5 467.0 ± 63.5

10 17.7 ± 1.5 145.3 ± 4.5 20.3 ± 5.1 445.0 ± 56.4 25 32.3 ± 9.0 137.0 ± 15.4 23.7 ± 3.1 480.0 ± 36.4 50 41.0 ± 4.4 145.3 ± 23.7 25.3 ± 4.0 477.0 ± 21.2

100 26.0 ± 3.6 P 141.0 ± 9.5 P 26.3 ± 1.5 P 556.7 ± 57.6 P 200 36.7 ± 8.3 P 142.0 ± 12.8 P 28.0 ± 11.3 P 473.3 ± 33.9 P

With S9 TMC278

400 34.0 ± 10.8 P 134.3 ± 22.9 P 20.7 ± 8.1 P 443.3 ± 42.8 P a unless stated otherwise; P: Precipitation; DMSO: dimethyl sulfoxide




Study TMC278-NC279: In Vitro Bacterial Reverse Mutation Test of TMC278 in Salmonella typhimurium with human liver S9-mix (continued)

Assay # 3 (Revertant Colony Counts (Mean ± SD)) Metabolic Activation Test Article Concentration

(μg/plate)a TA102 TA100

DMSO 0 244.0 ± 7.9 82.0 ± 3.6 Sodium Azide 2.5 - 1542.7 ± 25.8*

Without S9

4-Nitroquinoline-N-Oxide 5.00 1744.0 ± 85.1* - a unless stated otherwise; DMSO: dimethyl sulfoxide; -: Not Applicable; more than 2-fold increase TA100 and TA102 compared to the vehicle control. Assay # 3 (Revertant Colony Counts (Mean ± SD) Metabolic Activation Test Article Concentration

(μg/plate)a TA102

DMSO 0 273.3 ± 25.5 3 280.7 ± 33.2

7.81 372.7 ± 70.3 15.63 330.3 ± 43.0 31.25 314.0 ± 31.4 62.50 351.7 ± 17.0 125 380.3 ± 11.0 250 390.0 ± 13.2 P

With S9 TMC278

500 445.3 ± 24.8 P a unless stated otherwise; P: Precipitation; DMSO: dimethyl sulfoxide; -: Not Applicable.




Study TMC278-NC279: In Vitro Bacterial Reverse Mutation Test of TMC278 in Salmonella typhimurium with human liver S9-mix (continued)

Assay # 4 (Revertant Colony Counts (Mean ± SD)) Metabolic Activation Test Article Concentration

(μg/plate)a TA1537 TA100

DMSO 0 5.7 ± 2.3 71.3 ± 3.2 Sodium Azide 2.5 - 1435.3 ± 28.0*

Without S9

9-Aminoacridine 50.00 288.7 ± 38.7* - a unless stated otherwise; * more than 2-fold increase TA100 and more than 3-fold increase TA1537 compared to the vehicle control;

DMSO: dimethyl sulfoxide; -: Not Applicable. Assay # 4 (Revertant Colony Counts (Mean ± SD) Metabolic Activation Test Article Concentration

(μg/plate)a TA1537

DMSO 0 13.3 ± 2.3 3 13.0 ± 2.6

10 17.7 ± 2.5 25 10.3 ± 0.6 50 15.0 ± 4.4

100 17.3 ± 1.5 P 200 11.0 ± 5.3 P

With S9 TMC278

400 10.0 ± 2.6 P a unless stated otherwise; P: Precipitation, DMSO: dimethyl sulfoxide; -: Not Applicable

2.6.7.8.I. Genotoxicity: In Vitro Report Title: In Vitro Mammalian Forward Mutation Test with L5178Y Mouse Lymphoma Cells (TK- locus) using the Microtiter® Fluctuation Technique



2.6.7.8.I. TMC278-Exp5539: In Vitro Mammalian Forward Mutation Test with L5178Y Mouse Lymphoma Cells (TK-locus) using the Microtiter® Fluctuation Technique

Test: In Vitro Mammalian Forward Mutation Test Species/strain: L5178Y Mouse Lymphoma Cells (TK-locus) Metabolizing system: Aroclor 1254 induced rat liver S9 mix No. of independent assays: 5 No. of replicate cultures: 2 x 103cells/well

Treatment: Microtiter® Fluctuation Technique Concentrations: 0, 0.5, 1, 2, 2.5, 4, 5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 250, 500 g/mL TMC278 base Test article batch: Vehicle test article: DMSO Positive controls: DMN, MMS




Concentration (μg/mL)

Assay # 1

SG PE MF Total Relative Total Relative

RTG Total Corrected

DMSO 0 32 - 106 - - 84 74 1 37 114 98 92 105 - -

2.5 36 112 116 109 122 - - 5 36 112 91 85 95 - -

10 32 99 92 87 86 79 80 25 31 95 100 94 89 104 100 50 32 99 101 95 94 116 111

100 18 55 98 92 51 112 110 250* 12 36 80 76 27 121 147

TMC278

500* 7 21 82 77 16 94 109

3 hour treatment without S9

MMS 15 36 111 74 69 77 211 309 * precipitation, DMSO: dimethyl sulfoxide; - not applicable, SG: suspension growth, PE : plating efficiency, RTG: relative total growth, MF : mutant frequency, MMS: methyl

methanesulfonate, DMN: N-nitrosodimethylamine.




Study: TMC278-Exp5539: In Vitro Mammalian Forward Mutation Test with L5178Y Mouse Lymphoma Cells (TK- locus) using the Microtiter® Fluctuation Technique (continued)




Assay # 1


RTG Total Corrected

DMSO 0 31 - 95 - - 68 66 20 21 67 82 86 58 116 138 30 26 85 92 97 82 100 104 40 25 83 80 85 70 102 121 50 24 78 78 82 64 104 127 60 28 91 79 83 76 93 111 70* 26 84 78 82 69 111 137 80* 31 100 65 68 68 - - 90* 28 92 79 83 77 - -

TMC278

100* 26 84 61 65 55 - -


MMS 7.5 13 42 47 49 20 326 894 Assay # 2

DMSO 0 34 - 112 - - 72 60 3 hour treatment with S9 1 31 91 123 109 100 - -

2.5 36 104 138 124 128 - - 5 37 107 105 93 100 - - 10 36 106 112 100 106 106 91 15 36 106 95 85 90 110 112 20 32 95 123 109 104 92 71 25* 25 72 89 80 57 92 97 30* 29 86 106 95 82 91 81

TMC278

35* 18 54 114 102 55 114 97 DMN 400 nL/mL 35 103 82 73 75 259 366

*: precipitation, DMSO: dimethyl sulfoxide; - not applicable, SG : suspension growth, PE : plating efficiency, RTG: relative total growth, MF : mutant frequency, MMS: methyl methanesulfonate, DMN: N-nitrosodimethylamine




Study: TMC278-Exp5539: In Vitro Mammalian Forward Mutation Test with L5178Y Mouse Lymphoma Cells (TK- locus) using the Microtiter® Fluctuation Technique (continued)




Assay # 3


RTG Total Corrected

DMSO 0 34 - 110 - - 110 96 1 36 105 105 95 100 - -

2.5 34 98 114 104 102 104 87 5 34 100 91 82 82 98 103

10 38 109 98 89 97 106 104 25 31 91 94 85 78 138 146 50 27 77 95 86 67 124 128

100 12 35 94 85 29 110 113 250* 1 3 78 71 2 - -

TMC278

500* 1 2 60 54 1 - -


MMS 15 36 105 78 71 75 421 840 Assay # 4

DMSO 0 14 - 89 - - 131 144 2.5 17 120 89 100 120 - - 5 18 126 91 102 128 109 116

10 20 144 92 103 149 113 119 20 19 139 95 106 148 121 124 30 20 141 88 98 139 98 106 40 20 144 96 108 156 110 110 50* 20 143 85 96 136 122 139 60* 23 166 84 94 157 - -

TMC278

70* 22 161 89 100 161 - -


MMS 7.5 8 56 47 53 30 303 788 *: precipitation, DMSO: dimethyl sulfoxide; - not applicable, SG : suspension growth, PE : plating efficiency, RTG: relative total growth, MF : mutant frequency, MMS: methyl

methanesulfonate, DMN: N-nitrosodimethylamine.




Study: TMC278-Exp5539: In Vitro Mammalian Forward Mutation Test with L5178Y Mouse Lymphoma Cells (TK- locus) using the Microtiter® Fluctuation Technique (continued) Metabolic Activation



Assay # 5


RTG Total Corrected

DMSO 0 41 - 123 - - 99 77 0.5 47 115 106 87 100 - - 1 45 109 105 85 93 102 93 2 44 107 108 88 95 95 83 4 47 116 101 83 95 103 97 6 42 104 98 80 83 100 97 8* 36 88 100 81 72 95 90

10* 23 57 92 75 43 111 116 15* 0 0 - - - - -

TMC278

20* 0 0 - - - - -

3 hour treatment with S9

DMN 400 nL/mL 43 105 91 74 78 247 309 * precipitation, DMSO: dimethyl sulfoxide; - not applicable, SG : suspension growth, PE : plating efficiency, RTG: relative total growth, MF : mutant frequency, MMS: methyl

methanesulfonate., DMN: N-nitrosodimethylamine.

2.6.7.8.J. Genotoxicity: In Vitro Report Title: Mutation at the Thymidine Kinase (tk) Locus of Mouse Lymphoma L5178Y Cells (MLA) using the Microtitre® Fluctuation Technique



2.6.7.8.J. TMC278-NC336: Mutation at the Thymidine Kinase (tk) Locus of Mouse Lymphoma L5178Y Cells (MLA) using the Microtitre® Fluctuation Technique

Test: In Vitro Mammalian Forward Mutation Test Species/strain: L5178Y mouse lymphoma cells Metabolizing System: Aroclor 1254 induced rat liver S9 mix No of independent assays: 2 No of replicate cultures: 2

Treatment: Microtiter® Fluctuation Technique Concentrations: 0, 2.5, 5, 10, 12.5, 15, 20, 25, 30, 35, 40 and 50 g/mL TMC278.HCl Test article batch: Vehicle test article: DMSO Positive controls: MMS, B(a)P

Testing facility: Study no.: TMC278-NC336 GLP compliance: Yes

Assay # 1 Metabolic Activation



% RTG MF

DMSO 0 100 66.38 15 84 70.52 20 89 62.17 25 90 59.76 30* 74 63.29 40* 81 42.94

TMC278

50* 82 71.34 15 47 488.91

3 hours treatment without S9

MMS 20 32 571.54

DMSO 0 100 67.38 2.5 100 50.61 5 121 50.26

10 101 70.35 15 85 63.63

TMC278

20 87 47.75 2 47 673.77

3 hours treatment with S9

B(a)P 3 20 858.72

*: precipitation, DMSO: dimethyl sulfoxide; - not applicable, MMS: methyl methanesulfonate, MF : mutant frequency, RTG : relative total growth, B(a)P:benzo (a)pyrene




Study TMC278-NC336: Mutation at the Thymidine Kinase (tk) Locus of Mouse Lymphoma L5178Y Cells (MLA) using the Microtitre® Fluctuation Technique (continued) Assay # 2 Metabolic Activation



% RTG MF

DMSO 0 100 97.95 5 99 77.11

10 100 85.77 15 124 70.85 20* 142 87.89 25* 136 100.10

TMC278

30* 127 73.93 15 75 376.88


MMS 20 56 484.53

DMSO 0 100 64.48 2.5 93 68.65 5 83 68.72

10 82 56.94 12.5 76 68.65 15 70 70.99

TMC278

20* 72 86.05 2 15 983.93

3 hours treatment with S9

B(a)P 3 15 440.26 *: precipitation, DMSO: dimethyl sulfoxide; - not applicable, MMS: methyl methanesulfonate, MF : mutant frequency, RTG : relative total growth, B(a)P:benzo (a)pyrene




Study TMC278-NC336: Mutation at the Thymidine Kinase (tk) Locus of Mouse Lymphoma L5178Y Cells (MLA) using the Microtitre® Fluctuation Technique (continued) Assay # 2




% RTG MF

DMSO 0 100 104.54 2.5 98 76.96 5 95 82.34

10 67 76.78 20* 30 83.18 25* 52 100.66 30* 69 94.34 35* 85 83.53 40* 96 79.97

TMC278

50* 97 84.74 5 44 712.13


MMS 7.5 25 1210.66

* precipitation, DMSO : dimethyl sulfoxide; - : not applicable, MMS : methyl methanesulfonate, MF : mutant frequency, RTG : relative total growth.

2.6.7.8.K. Genotoxicity: In Vitro Report Title: Mutagenicity Test with GS-4331-05 in the Salmonella-Escherichia Coli Mammalian Microsome Reverse Mutation Assay


**TA98 2-aminoanthracene 2.5 g/plate ***TA98 2-nitrofluorene 1.0 g/plate TA1535 2-aminoanthracene 2.5 g/plate TA1535 sodium azide 2.0 g/plate TA1537 2-aminoanthracene 2.5 g/plate TA1537 ICR-191 2.0 g/plate WP2uvrA 2-aminoanthracene 25.0 g/plate WP2uvrA 4-nitroquinoline-N-oxide 1.0 g/plate TA100 was plated as part of the experiment. However, due to confluent bacterial growth (contamination) on the plates, no data were generated. Background lawn normal


2.6.7.8.K. 96-TOX-4331-005: Mutagenicity Test with GS-4331-05 in the Salmonella-Escherichia Coli Mammalian Microsome Reverse Mutation Assay

Test for induction of : Reverse mutations No of independent assays (trials) : 3 Study No: 96-TOX-4331-005 Strains: S. typhimurium strains TA98, TA100, TA1535, TA1537 & E. Coli strain WP2uvrA

No of replicate cultures: 3 per test concentrations, vehicle controls & positive controls

Metabolising system: AroclorTM -induced rat liver (S9) No of cells analysed culture: - GLP compliance: Yes Vehicles: Test Article: DMSO Positive control: DMSO Date of treatment: 19 (study completed) Treatment: Plate incorporation for 48hr ± 8 hr. Cytotoxic effects: None Genotoxic effects: The only positive result was a 3.2 fold increase above control value in the number of revertants per plate in 1 of 3 assays (2.4, 3.2 & 1.2 fold in the respective assays) with strain TA1535 in the absence of S9. No other mutagenicity was seen. All positive controls in each replicate and in each assay evidenced mutagenic activity. Assay 1: Assay ID 18133-B1

MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION DOSE/PLATE TA98 TA1535 TA1537 WP2uvrA (μg) MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. Microsomes: Rat Liver

Vehicle Control - 29 2 15 3 24 3 17 2 Test Article 100 25 4 16 2 15 3 15 4

333 24 1 32 12 14 2 14 5 1000 28 2 36 7 25 9 19 6 3330 32 3 36 4 18 4 15 4 5000 32 3 36 3 21 2 18 3

Positive Control ** 942 50 183 13 148 74 374 79 Microsomes: None

Vehicle Control - 17 3 12 4 16 5 13 1 Test Article 100 15 6 12 3 20 5 16 4 333 19 2 11 2 17 3 16 4 1000 14 5 21 5 15 3 15 5 3330 18 5 24 4 14 2 13 2 5000 15 6 29 4 11 3 15 6 Positive Control *** 155 13 622 71 716 116 197 45



** TA100 2-aminoanthracene 2.5 g/plate

*** TA100 sodium azide 2.0 g/plate TA1535 2-aminoanthracene 2.5 g/plate TA1535 sodium azide 2.0 g/plate TA1537 2-aminoanthracene 2.5 g/plate A1537 ICR-191 2.0 g/plate Background lawn normal CONFIDENTIAL Page 195 17AUG2010

Assay 2: Assay ID 18133-B2 MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION

DOSE/PLATE TA100 TA1535 TA1537 ( g) MEAN S.D. MEAN S.D. MEAN S.D. Microsomes: Rat Liver

Vehicle Control - 147 1 9 0 11 6 Test Article 100 127 2 16 4 10 5 333 141 5 15 3 8 1 1000 147 8 15 3 9 2 3330 196 32 13 3 11 4 5000 184 21 23 8 10 3 Positive Control ** 1098 164 124 14 127 26

Microsomes: None Vehicle Control - 80 16 11 3 7 3 Test Article 100 76 17 13 5 4 1 333 77 17 18 0 4 1 1000 90 13 19 6 4 2 3330 109 11 35 11 8 3 5000 90 7 35 9 8 2 Positive Control *** 430 32 511 61 93 24



** TA1535 2-aminoanthracene 2.5 g/plate ..*** TA1535 sodium azide 2.0 g/plate TA1537 2-aminoanthracene 2.5 g/plate TA1537 ICR-191 2.0 g/plate Background lawn normal CONFIDENTIAL Page 196 17AUG2010

Assay B: Assay ID 18133-B3 MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION

DOSE/PLATE TA1535 TA1537 ( g) MEAN S.D. MEAN S.D. Microsomes: Rat Liver

Vehicle Control: - 14 3 9 1 Test Article: 100 14 2 7 5 333 17 5 9 4 1000 19 3 8 1 3330 22 2 8 1 5000 19 3 6 5 Positive Control: ** 142 7 223 17

Microsomes: None Vehicle Control: - 24 4 4 1 Test Article: 100 20 4 7 2 333 21 1 7 3 1000 26 5 7 5 3330 28 1 8 6 5000 24 9 2 2 Positive Control *** 740 18 1714 138

2.6.7.8.L. Genotoxicity: In Vitro Report Title: Mutagenicity test with GS-1278 (PMPA) Lot No. , GS-1278 (PMPA) Lot No. , GS-4331-05 (PMPA prodrug, bis-POC PMPA) Lot No. ( ) in the Salmonella/Mammalian-Microsome Reverse Mutation Assay


** TA1535 2-aminoanthracene 2.5 g/plate Background lawn normal CONFIDENTIAL Page 197 17AUG2010

2.6.7.8.L. 97-TOX-1278-003: Mutagenicity Test with GS-1278 (PMPA) Lot No. , GS-1278 (PMPA) Lot No. , GS-4331-05 (PMPA prodrug, bis-POC PMPA) Lot No. ( ) in the Salmonella/Mammalian-Microsome Reverse Mutation Assay

Test for Induction of : Reverse mutations No of Independent Assays (trials) : 3 Study No: 97-TOX-1278-003 Strains: S. typhimurium strains TA1535 No of Replicate Cultures: 3 per test concentrations,

vehicle controls & positive controls

Metabolising System: AroclorTM -induced rat liver (S9) No of Cells Analysed Culture: - GLP Compliance: Yes Vehicles: Test Article: DMSO (TDF), deionised water (tenofovir) Positive control: See footer Date of Treatment: 19 (study completed) Treatment: Plate incorporation for 52hr ± 4 hr. Cytotoxic Effects: - Genotoxic Effects: Under the conditions of the study, TDF & tenofovir did not cause any positive increases in the number of revertants per plate with tester strain TA1535 in the presence of microsomal enzymes prepared from rat liver (S9). The positive control in each assay and in each experiment evidenced mutagenic activity. Assay 1: Assay ID 19115-B1

REVERTANTS PER PLATE DOSE/PLATE TA1535 TA1535

( g) 1 2 3 MEAN S.D. Microsomes: Rat Liver

Vehicle Control: - 11 15 17 14 3 Test Article: 100 7 10 15 11 4 (Tenofovir, 333 12 17 18 16 3 Lot No. ) 667 9 11 12 11 2 1000 10 13 20 14 5 3330 23 25 30 26 4 5000 31 33 37 34 3 Positive Control ** 129 150 175 151 23



** TA1535 2-aminoanthracene 2.5 g/plate Background lawn normal CONFIDENTIAL Page 198 17AUG2010

Assay 2: Assay ID 19116-B1 REVERTANTS PER PLATE

DOSE/PLATE TA1535 TA1535


Vehicle Control: - 7 11 13 10 3 Test Article: 100 9 10 12 10 2 (Tenofovir, 333 8 9 9 9 1 Lot No. ) 667 9 10 11 10 1 1000 7 7 14 9 4 3330 15 16 23 18 4 5000 13 13 17 14 2 Positive Control: ** 91 100 131 107 21



** TA1535 2-aminoanthracene 2.5 g/plate C = no count due to contamination on the plate. Background lawn normal CONFIDENTIAL Page 199 17AUG2010

Assay 3: Assay ID 19117-B1 REVERTANTS PER PLATE

DOSE/PLATE TA1535 TA1535


Vehicle Control: - C 9 11 10 1 Test Article: 100 6 12 16 11 5 (TDF, 333 9 12 17 13 4 Lot No. 667 4 5 7 5 2 ( ) 1000 7 13 17 12 5 3330 11 14 15 13 2 5000 7 13 22 14 8 Positive Control: ** 101 94 102 99 4

2.6.7.8.M. Genotoxicity: In Vitro Report Title: Mutagenicity Test of Tenofovir DF Using Microorganisms



2.6.7.8.M. K01-3037: Mutagenicity Test of Tenofovir DF Using Microorganisms

Test for Induction of: Reverse mutation in bacterial cells No. of Independent Assays: 3 Study No.: K01-3037 Species/Strain: S. typhimurium strains TA98, TA100,

TA1535, TA1537 & E. Coli strain WP2uvrA

No. of Replicate Cultures: 3 (all negative controls), 2 (#1), 2 (#2), 2 (#3)

Metabolizing System: Phenobarbital and 5, 6-benzoflavone induced rat liver S9

GLP Compliance: Yes

Vehicles: For Test Article: Dehydrated DMSO For Positive Controls: Dehydrated DMSO, except distilled water for sodium azide Treatments: Pre-incubation method for initial (#1) and confirmatory (#2, #3) assays Date of Treatments: 20 ,

20 and 20

Cytotoxic Effects: No appreciable toxicity was observed. Genotoxic Effects: None

Metabolic Dose Level Assay No. 1 Initial Mutagenicity Assay

Revertant Colony Counts (Mean) Activation Test Article ( g/plate) TA100 TA1535 WP2 uvr A TA98 TA1537 Without (Vehicle) -- 142 14 24 25 14 Activation Test Article 4.88 145 15 24 19 6 19.5 152 10 19 21 12 78.1 167 10 24 26 10 313 156 15 25 17 11 1250 156 16 21 21 13 5000 176 26 26 26 11 AF-2 0.01 631 173 Sodium azide 0.5 491 AF-2 0.1 486 ICR-191 0.5 962




Metabolic Dose Level Assay No. 1 Initial Mutagenicity Assay

Revertant Colony Counts (Mean) Activation Test Article ( g/plate) TA100 TA1535 WP2 uvr A TA98 TA1537 With (Vehicle) -- 151 10 23 28 23 Activation Test Article 4.88 182 8 27 30 20 19.5 164 11 25 29 25 78.1 168 13 26 47 29 313 179 16 33 37 30 1250 195 12 34 52 26 5000 236 8 31 56 21 2AA 1 1004 2AA 2 241 221 2AA 10 592 2AA 0.5 415

Metabolic Dose Level Assay No. 2 Confirmatory Mutagenicity Assay

Revertant Colony Counts (Mean) Activation Test Article ( g/plate) TA100 TA1535 WP2 uvr A TA98 TA1537 Without (Vehicle) -- 169 15 34 22 14 Activation Test Article 156 161 17 32 26 13 313 167 12 39 19 17 625 149 16 35 18 16 1250 154 18 42 25 15 2500 184 20 32 29 10 5000 179 22 37 21 12 AF-2 0.01 653 210 Sodium azide 0.5 460 AF-2 0.1 507 ICR-191 0.5 1149





Revertant Colony Counts (Mean) Activation Test Article ( g/plate) TA100 TA1535 WP2 uvr A TA98 TA1537 With (Vehicle) -- 168 9 41 30 24 Activation Test Article 156 175 7 41 36 22 313 186 10 32 34 21 625 204 12 38 43 27 1250 211 10 44 46 27 2500 246 10 48 55 25 5000 253 14 46 51 26 2AA 1 958 2AA 2 263 233 2AA 10 650 2AA 0.5 422


Revertant Colony Counts (Mean) Activation Test Article ( g/plate) TA98 Without (Vehicle) -- 30 Activation AF-2 0.1 463 With (Vehicle) -- 42 Activation Test Article 156 37 313 53 625 45 1250 47 2500 52 5000 51 2AA 0.5 423

2.6.7.8.N. Genotoxicity: In Vitro Report Title: Mutagenicity Test on GS-4331-05 in the L5178Y TK “+/-” Mouse Lymphoma Forward Mutation Assay



2.6.7.8.N. 97-TOX-4331-007: Mutagenicity Test on GS-4331-05 in the L5178Y TK “+/-” Mouse Lymphoma Forward Mutation Assay

Test for Induction of: Forward mutations No of Independent Assays (trials) : 3 Study No: 97-TOX-4331-007 Strains: Mouse lymphoma L5178Y cell line No of Replicate Cultures: 3 in vehicle & 1 each

positive controls & treatments

Metabolising System: AroclorTM -induced rat liver (S9) No of Cells Analysed Culture: - GLP Compliance: Yes Vehicles: Test Article: DMSO Positive Control: DMSO Date of Treatment: 19 (study

completed) Treatment: Cytotoxicity dose ranging test: 4 hr treatment period, Mutation assay 4 hr treatment period Cytotoxic Effects: Cytotoxicity assay: The measure of cytotoxicity of each treatment was the relative suspension of growth of cells over the 2 day expression period multiplied by the relative cloning efficiency at the time of selection (percent relative growth). A dose range-finding study tested the vehicle and TDF 9.85 to 5000 g/mL metabolic activation (S9). Under nonactivation, TDF was noncytotoxic to weakly cytotoxic from 9.85 to 78.5 g/mL, highly cytotoxic at 157 g/mL, and excessively toxic or lethal at higher levels. With activation, TDF was noncytotoxic to weakly cytotoxic from 9.85 to 78.5 g/mL, and excessively toxic or lethal at higher levels. Nonactivation Mutation assay: TDF was excessively cytotoxic above 125 g/mL (higher concentrations were not plated for selection). After cloning for mutant analysis, 125 g/mL was excessively cytotoxic. The remaining treatments (12.5–100 g/mL) induced cytotoxicity ranging from 15.6 % to 89.1% relative growths. Metabolic Activation Mutation assay: TDF treatments above 150 g/mL were terminated because of excessive toxicity. After cloning for mutant analysis, 125 g/mL and 150 g/mL were excessively cytotoxic. The remaining treatments (12.5–100 g/mL) were noncytotoxic to highly cytotoxic (13.3% to 105.6% relative growths). Genotoxic effects: TDF was positive with and without S9 metabolic activation in L5178Y TK+/- mouse lymphoma forward mutation assay over a wide range of cytotoxicity. In the nonactivation assay, dose related increases (3.1 to 16.4-fold) in the mutant frequency were observed from 50 to 100 g/mL. In the activation assay, dose related increases (5.0 to 8.6-fold) in the mutant frequency were observed from 50 to 100 g/mL. Mutant colonies from the nonactivation and nonactivation assays treated with test material demonstrated a preferential increase in small colonies, suggesting chromosomal damage. Effects of the positive control : Both positive control articles (MMS and MMC) demonstrated both large and small colonies. Both (with and without activation) induced large increases in mutant frequency in excess of minimum criteria.

2.6.7.8.N. Genotoxicity: In Vitro Report Title: Mutagenicity test on GS-4331-05 in the L5178Y TK “+/-” Mouse Lymphoma Forward Mutation Assay


a Cell density determined by hemocytometer approximately 24 hours after treatment initiation. b Relative to vehicle control cell density for all treatments c VC = Vehicle control, 1.0% DMSO CONFIDENTIAL Page 204 17AUG2010

CYTOTOXICITY ASSAY Four-Hour Treatment Period Applied TDF Concentration Without S9 Activation

Cell Density/ml % Vehicle With S9 Activation

Cell Density/mL % Vehicle g/mL (x105)a Controlb (x105)a Controlb VCc 11.6 100.0 7.8 100.0 9.85 11.8 101.7 9.3 119.2 19.7 11.3 97.4 7.3 93.6 39.3 10.9 94.0 7.4 94.9 78.5 5.4 46.6 4.4 56.4 157 1.3 11.2 0.7 9.0 313 0.2 1.7 0.3 3.8 625 0.0 0.0 0.0 0.0

1250 0.0 0.0 0.0 0.0 2500 0.0 0.0 0.0 0.0 5000 0.0 0.0 0.0 0.0



a suspension growth = (day 1 count/3) * (day 2 count) /3 (or day 1 count if not split back) b cloning efficiency = total viable colony count / number of cells seeded * 100 c relative growth = (relative suspension growth * relative cloning efficiency) / 100 d mutant frequency = (total mutant colonies / total viable colonies) x 2x10-4. decimal is moved to express the frequency in units of 10-6 e vehicle control = 1% dms0. mms = methanesulfonate positive control. mca = methylcholanthrene positive control f mutagenic - exceeds minimum criterion of 84.9 x 10-6 CONFIDENTIAL Page 205 17AUG2010

MUTATION ASSAY WITHOUT ACTIVATION

Test Condition

Daily Cell Counts (Cells/mL. 105 units)

Suspension Growth

Avg Vehicle Control

Total Mutant Colonies

Total Viable

Colonies Cloning

Efficiency

Avg Vehicle Control

Relative Growth

(%)c

Mutant Frequency (10-6 units)d

1 2 Non Activation Controls e

Vehicle Control 14.8 13.2 21.7 96 517 86.2 100.0 37.1 Vehicle Control 11.4 15.9 20.1 119 492 82.0 100.0 48.4 Vehicle Control 12.3 13.0 17.8 19.9 112 536 89.3 85.8 100.0 41.8 MMS 5 nl/mL 7.8 14.3 12.4 619 408 68.0 49.4 303.4f MMS 10 nl/mL 8.3 12.9 11.9 595 193 32.2 22.4 616.6f TDF ( g/mL)

Relative to Vehicle

Control (%)

Relative to Vehicle Control

(%)

12.5 11.2 15.7 98.2 92 467 90.7 89.1 39.4 25.0 11.0 15.3 94.0 85 424 82.4 77.5 40.1 50.0 11.0 11.7 71.9 330 505 98.1 70.5 130.7f 62.5 8.3 14.3 66.3 578 385 74.8 49.6 300.3f 75.0 10.0 10.8 60.3 721 404 78.5 47.3 356.9f 100 5.4 8.8 26.5 1048 302 58.7 15.6 694.0 f 125 2.9* 8.6 14.4 644 147 28.6 4.1 876.2 f



a suspension growth = (day 1 count/3) * (day 2 count) /3 (or day 1 count if not split back) b cloning efficiency = total viable colony count / number of cells seeded * 100 c relative growth = (relative suspension growth * relative cloning efficiency) / 100 d mutant frequency = (total mutant colonies / total viable colonies) x 2x10-4. decimal is moved to express the frequency in units of 10-6 e vehicle control = 1% dms0. mms = methanesulfonate positive control. mca = methylcholanthrene positive control f mutagenic - exceeds minimum criterion of 84.9 x 10-6 CONFIDENTIAL Page 206 17AUG2010

MUTATION ASSAY WITH ACTIVATION

Test Condition Daily Cell Counts

(Cells/mL. 105 units Suspension

Growth

Avg Vehicle Control

Total Mutant Colonies

Total Viable Colonies

Cloning Efficiency

Avg Vehicle Control

Relative Growth

(%)c

Mutant Frequency (10-6 units)d

1 2 S9 Activation Controls e

Vehicle Control 9.6 14.1 15.0 187 540 90.0 100.0 69.3 Vehicle Control 9.3 15.9 16.4 140 523 87.2 100.0 53.5 Vehicle Control 10.8 13.8 16.6 16.0 138 517 86.2 87.8 100.0 53.4 MCA 2 l/mL 6.4 16.1 11.4 901 574 95.7 77.7 313.9f MCA 4 l/mL 5.3 16.8 9.9 887 608 101.3 71.4 291.8 f TDF (μg/mL)

Relative to Vehicle

Control (%)

Relative to Vehicle

Control (%)

12.5 7.4 16.4 84.3 211 419 79.5 67.0 100.7 25.0 8.9 16.2 100.1 308 556 105.5 105.6 110.8 50.0 8.1 11.1 62.4 708 486 92.3 57.6 291.4 f 62.5 7.4 10.5 54.0 782 509 96.6 52.2 307.3 f 75.0 3.7* 17.0 35.4 798 373 70.8 25.1 427.9 f 100 3.4* 13.4 27.9 628 250 47.5 13.3 502.4 f 125 2.3* 8.3 17.3 668 225 42.7 7.4 593.8 f 150 1.0* 4.4 9.2 755 276 52.4 4.8 547.1 f

2.6.7.8.O. Genotoxicity: In Vitro Report Title: Bacterial Reverse Mutation Assay with with Emtricitabine/Tenofovir Disoproxil Fumarate

Test Article: Emtricitabine/Tenofovir Disoproxil Fumarate


2.6.7.8.O. TX-164-2002: Bacterial Reverse Mutation Assay with Emtricitabine/Tenofovir Disproxil Fumarate

Report Title: Bacterial Reverse Mutation Assay Test for Induction of: Reverse mutation in bacterial

cells No. of Independent Assays:

2 Study No.: TX-164-2002

Species/Strain: S. typhimurium, E. coli No. of Replicate Cultures:

2 (#1), 3 (#2)

Metabolizing System: Aroclor-induced rat liver S9 GLP Compliance: Yes Vehicles: For Test

Article: DMSO For Positive

Controls: DMSO, except water for sodium azide

Treatments: Plate incorporation for initial (#1) and confirmatory (#2) assays Date of Treatments: 20 and 20

Cytotoxic Effects: No appreciable toxicity was observed. Genotoxic Effects: None

Metabolic Dose Level Assay No. 1 Initial Mutagenicity Assay Revertant Colony Counts (Mean SD )

Activation Test Article ( g/plate) TA98 TA100 TA1535 TA1537 WP2 uvrA Without (Vehicle) 50 L/plate 13 ± 4 112 ± 12 19 ± 6 5 ± 1 16 ± 4 Activation Test Article 1.5 13 ± 1 149 ± 16 21 ± 5 5 ± 1 13 ± 4 5.0 12 ± 1 135 ± 14 17 ± 4 6 ± 1 14 ± 2 15 15 ± 1 111 ± 8 15 ± 0 5 ± 2 12 ± 4 50 17 ± 1 127 ± 5 16 ± 1 7 ± 3 13 ± 1 150 13 ± 3 144 ± 16 16 ± 4 8 ± 1 19 ± 1 500 15 ± 3 135 ± 16 16 ± 6 5 ± 1 14 ± 3 1500 13 ± 3 157 ± 3 17 ± 3 6 ± 1 14 ± 2 5000 11 ± 0 155 ± 6 16 ± 7 7 ± 1 12 ± 3 2-nitrofluorene 1.0 170 ± 11 Sodium azide 1.0 546 ± 11 398 ± 6 9-aminoacridine 75 1424 ± 127 MMS 1000 128 ± 14




Metabolic Dose Level Assay No. 1 Initial Mutagenicity Assay Revertant Colony Counts (Mean SD )

Activation Test Article ( g/plate) TA98 TA100 TA1535 TA1537 WP2 uvrA With (Vehicle) 50 L/plate 16 ± 1 143 ± 11 14 ± 8 9 ± 2 15 ± 0 Activation Test Article 1.5 20 ± 8 131 ± 25 14 ± 1 7 ± 1 13 ± 1 5.0 17 ± 1 146 ± 5 10 ± 3 8 ± 4 15 ± 2 15 18 ± 2 143 ± 21 11 ± 1 10 ± 2 16 ± 1 50 22 ± 8 127 ± 6 15 ± 4 8 ± 1 22 ± 4 150 18 ± 1 140 ± 1 15 ± 1 6 ± 1 18 ± 2 500 19 ± 4 148 ± 31 12 ± 4 7 ± 1 14 ± 4 1500 22 ± 4 148 ± 9 11 ± 1 8 ± 0 11 ± 3 5000 20 ± 1 127 ± 4 13 ± 1 10 ± 1 17 ± 6 2-aminoanthracene 1.0 689 ± 145 734 ± 0 107 ± 28 93 ± 33 2-aminoanthracene 10 608 ± 76





Revertant Colony Counts (Mean SD ) Activation Test Article ( g/plate) TA98 TA100 TA1535 TA1537 WP2 uvrA Without (Vehicle) 50 L/plate 22 ± 3 109 ± 22 16 ± 2 6 ± 2 12 ± 4 Activation Test Article 50 27 ± 2 147 ± 3 19 ± 4 6 ± 4 12 ± 2 150 29 ± 6 104 ± 23 14 ± 2 5 ± 1 13 ± 4 500 32 ± 6 142 ± 6 18 ± 3 6 ± 2 13 ± 2 1500 29 ± 3 127 ± 3 17 ± 2 4 ± 1 14 ± 3 5000 28 ± 4 132 ± 25 22 ± 4 5 ± 3 10 ± 4 2-nitrofluorene 1.0 173 ± 23 Sodium azide 1.0 569 ± 17 357 ± 20 9-aminoacridine 75 1282 ± 126 MMS 1000 111 ± 13 With (Vehicle) 50 L/plate 26 ± 5 136 ± 5 15 ± 3 4 ± 1 11 ± 4 Activation Test Article 50 30 ± 4 139 ± 14 12 ± 4 6 ± 2 14 ± 1 150 32 ± 2 143 ± 8 10 ± 3 5 ± 2 11 ± 1 500 28 ± 3 159 ± 21 13 ± 5 7 ± 4 11 ± 4 1500 30 ± 3 150 ± 9 13 ± 3 5 ± 3 14 ± 4 5000 27 ± 3 130 ± 19 13 ± 1 10 ± 2 11 ± 3 2-aminoanthracene 1.0 640 ± 75 917 ± 64 101 ± 13 79 ± 17 2-aminoanthracene 10 581 ± 82

2.6.7.8.P. Genotoxicity: In Vitro Report Title: In Vitro Mammalian Cell Gene Mutation Test (L5178Y/TK+/- Mouse Lymphoma Assay) with Emtricitabine/Tenofovir Disoproxil Fumarate

Test Article: Emtricitabine (FTC) / Tenofovir Disoproxil Fumarate (TDF)


2.6.7.8.P. TX-164-2003: In Vitro Mammalian Cell Gene Mutation Test (L5178Y/TK+/- Mouse Lymphoma Assay) with Emtricitabine/Tenofovir Disoproxil Fumarate

Test for induction of : Forward mutations at the thymidine kinase locus in cultured L5178Y cells. Mutants are unable to metabolize the non-toxic thymidine metabolite, trifluorothymidine (TFT) to its lethal metabolite and hence are resistant to its lethal effect.

No of independent assays : Preliminary Toxicity assay: 1 experiment Mutagenicity assay: 1 experiment

Study No: AA97YC-YD.704.BTL Sponsor Study No.: TX-164-2003

Strains: L5178Y mouse lymphoma cells No. of replicate cultures1:

Preliminary toxicity assay: a) single, b) single Mutagenicity assay: a) duplicate, single for positive controls, b) duplicate, single for positive controls

Metabolizing system: Aroclor 1254-induced rat liver (S9 mix)

No. of cells analyzed / culture: Adjusted to 3 x 105/mL at 24 and 48 hours after treatment. Cultures less than 3 x 105/mL were not adjusted

Test Article Vehicle: Dimethyl sulfoxide (DMSO)

Positive control: a) –S9: methyl methanesultonate (MMS) 15 and 20 g/mL, b) +S9: 7,12-dimethyl-benz(a)anthracene) 2.5 and 4.0 g/mL

GLP compliance: Yes

Treatment: Treatment period was 4 hours with and without S9 activation at 37±1ºC. Recovery at approximately 24 and 48 hours after treatment (+/-S9)

Dates of treatment: Preliminary toxicity assay: 20 Mutagenicity assay: 20

Cytotoxic effects: Suspension growth relative to the solvent controls at 3575 g/mL (10 mM) was 3% without activation and 0% with S9 activation in the Mouse Lymphoma Mutagenesis Assay. Genotoxic effects: FTC (Emtricitabine)/TDF (Tenofovir Disoproxil Fumarate) was concluded to be positive in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.

1 Preliminary toxicity assay a) –S9: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 3575 g/mL. Vehicle control. b) +S9: (same) Mutagenicity assay a) –S9: 15, 50, 100, 150, 200, 300 g/mL. Positive and vehicle controls. b) +S9: 50, 100, 200, 300, 400, 500, g/mL. Positive and vehicle controls.




Assay 1: MUTATION ASSAY WITHOUT ACTIVATION

Test Condition Daily cell counts (Conc. 106 cells)a

Mutant colonies avg.

Suspension growth totalb

Suspension growth

control (%)c

Viable colonies

(VC) avg.

Cloning growth

control (%)d

Total growth

(%)e

Mutant frequency (106 cells)f

Day 1 Day 2 Vehicle control

1 1.366 1.688 78 25.6 221 71 2 1.386 1.654 68 25.5 173 78

Positive control: Methyl methanesulfonate ( g/mL) 15 1.194 1.590 188 21.1 83 150 76 63 251 20 1.154 1.726 205 22.1 87 162 83 72 253

FTC/TDF ( g/mL) 15 A 1.265 1.563 53 22.0 86 191 97 84 56 15 B 1.266 1.799 63 25.3 99 188 96 95 67 50 A 1.214 1.735 69 23.4 92 183 93 85 75 50 B 1.307 1.709 69 24.8 97 165 84 82 84

100 A 1.128 1.691 128 21.2 83 154 78 65 167 100 B 1.159 1.651 134 21.3 83 69 35 29 386 150 A 0.823 1.466 289 13.4 52 97 49 26 595 150 B 0.857 1.461 257 13.9 54 109 55 30 474 200 A 0.592 0.980 ++ 6.4 25 66 33 8 ++ 200 B 0.612 0.966 247 6.6 26 84 43 11 591

++ - Too toxic to count, total growth <10% a - Cultures containing <0.3x106 cells/mL on day 1 and 2 are considered to have 0% total suspension growth. b - Total suspension growth = Day 1 cell conc. x Day 2 cell conc. 0.3x106 cells/mL Day 1 adjusted cell conc. c - % of control suspension growth = total treatment suspension growth x 100 average solvent control total suspension growth d - % control cloning growth = average VC of treated culture x 100 average VC of solvent control e - % total growth = (% suspension growth)(% cloning growth) 100 f - Mutant frequency per 106 surviving cells = Average # TFT colonies x 200 average # VC colonies




Assay 1: MUTATION ASSAY WITH ACTIVATION

Test Condition Daily cell counts (Conc. 106 cells)a

Mutant colonies avg.

Suspension growth totalb

Suspension growth

control (%)c

Viable colonies

(VC) avg.

Cloning growth

control (%)d

Total growth

(%)e

Mutant frequency (106 cells)f

Day 1 Day 2 Vehicle control

1 0.953 1.462 94 15.5 196 96 2 0.983 1.458 84 15.9 169 100

Positive control: 7,12 Dimethylbenz(a)anthracene ( g/mL) 2.5 0.770 1.489 229 12.7 81 112 61 50 411 4.0 0.600 1.074 249 7.2 46 77 42 19 650

FTC/TDF ( g/mL) 50 A 0.890 1.499 141 14.8 94 159 87 82 177 50 B 0.919 1.455 115 14.9 95 158 86 82 146

100 A 0.850 1.598 171 15.1 96 167 91 88 205 100 B 0.846 1.561 176 14.7 93 121 66 62 291 200 A 0.738 1.596 149 13.1 83 141 77 64 211 200 B 0.800 1.628 122 14.5 92 140 77 71 174 300 A 0.592 1.408 197 9.3 59 125 68 40 316 300 B 0.593 1.441 179 9.5 60 129 71 43 277 400 A 0.339 1.133 201 4.3 27 91 50 14 443 400 B 0.345 1.167 227 4.5 28 102 56 16 447

++ - Too toxic to count, total growth <10% a - Cultures containing <0.3x106 cells/mL on day 1 and 2 are considered to have 0% total suspension growth. b - Total suspension growth = Day 1 cell conc. x Day 2 cell conc. 0.3x106 cells/mL Day 1 adjusted cell conc. c - % of control suspension growth = total treatment suspension growth x 100 average solvent control total suspension growth d - % control cloning growth = average VC of treated culture x 100 average VC of solvent control e - % total growth = (% suspension growth)(% cloning growth) 100 f - Mutant frequency per 106 surviving cells = Average # TFT colonies x 200 average # VC colonies

2.6.7.9.A. Genotoxicity: In Vivo Report Title: In Vivo Mammalian Erythrocyte Micronucleus Test



2.6.7.9. Genotoxicity (In Vivo) 2.6.7.9.A. TOX011: In Vivo Mammalian Erythrocyte Micronucleus Test

Test for Induction of: Micronucleus formation Treatment Schedule: Single dose 500, 1000 & 2000 mg/kg

Study No: TOX011

Strains: Mouse / ICR Sampling Time: 24 hours and 48 hours (vehicle control and high test dose 2000 mg/kg only)

Age: 6 to 8 weeks old Method of Administration: Oral gavage Cells Evaluated: Polychromatic erythrocytes in bone marrow Vehicle/Formulation: Sterile distilled water

10 mL/kg + FTC (free base) GLP Compliance: Yes

No. of Cells Analyzed/Animal: Bone marrow was collected from femurs and 2 to 4 slides were prepared from each mouse. The number of micronucleated normochromic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated.

Date of Dosing: , 19 – , 19

Special Features: none Toxic/Cytotoxic Effects: No mortality, clinical signs of toxicity, or evidence of bone marrow toxicity were observed in any mice in any emtricitabine treated group during the course of the micronucleus study. All mice appeared normal throughout the observation period. Genotoxic Effects: Emtricitabine did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male ICR mice.

2.6.7.9.A. Genotoxicity: In Vivo Report Title: In Vivo Mammalian Erythrocyte Micronucleus Test



Test Article Dose

(mg/kg) No. of

Animals Harvest Time

(hour) PCE/Total Erythrocytes

(mean sd) Number/1000 PCEs

(mean ± sd) Number/PCEs

Scored

Vehicle control (water) Vehicle 5 24 0.60 0.01 0.8 ± 0.27 8/10,000

Vehicle 5 48 0.59 0.05 0.4 ± 0.42 4/10,000

Positive control (Cyclophosphamide)

5 24 0.57 0.02 40.0 ± 6.62 400/10,000*

Test Article

Low test dose 500 5 24 0.61 0.02 0.5 ± 0.35 5/10,000

Mid test dose 1000 5 24 0.56 0.06 1.1 ± 0.55 11/10,000

High test dose 2000 5 24 0.58 ± 0.03 1.2 ± 1.52 12/10,000

2000 5 48 0.57 ± 0.05 0.2 ± 0.27 2/10,000

PCE = Polychromatic Erythrocytes * p 0.05, ANOVA followed by Dunnett’s t-test

2.6.7.9.B. Genotoxicity: In Vivo Report Title: In Vivo Micronucleus test on Bone Marrow Cells of Mice



2.6.7.9.B. TMC278-Exp5538: In Vivo Micronucleus Test on Bone Marrow Cells of Mice

Test: In vivo micronucleus test Species/strain: Mouse/SPF Albino Swiss (CD1) Age at first dose: Approximately 8 weeks Cells evaluated: PCEs of bone marrow No. of cells analyzed/animal: 1000 (PCEs + NCEs) to determine bone marrow proliferation and 2000 PCEs to screen for micronuclei

Treatment: polyethylene glycol (PEG) 400 solution with 100 mg/ml citric acid Administration route/method: oral/gavage Doses: 0, 100, 400 and 1600 mg/kg b.w TMC278 base Test article batch: Vehicle/formulation: Polyethylene glycol 400 solution with 100 mg/mL citric acid


Toxic/Cytotoxic effects: no statistically significant bone marrow toxic effects were observed following treatment with 100, 400 or 1600 mg/kg TMC278 base. Genotoxic effects: no genotoxic effects were observed following treatment with 100, 400 or 1600 mg/kg TMC278. under the conditions described in this test Dose (mg/kg/day) 0 100 400 1600 No. of animals M:5 F:5 M:5 F:5 M:5 F:5 M:5 F:5 Died or killed prematurely 0 0 0 0 0 0 0 0

0 100 400 1600 Dose (mg/kg/day) No. of animals - - M:6 F:6 M:6 F:6 M:10 F:10 C1h ( g/mL) - - 39 33 59 68 60 58 AUC0-6h Day 1 ( g.h/mL) - - 158 130 262 258 307 287

2.6.7.9.B. Genotoxicity: In Vivo Report Title: In Vivo Micronucleus test on Bone Marrow Cells of Mice



Study: TMC278-Exp5538: In vivo Micronucleus Test on Bone Marrow Cells of Mice (continued)

Noteworthy Findings Control, Test and Reference Articles

Dose (mg/kg body weight)

Number of Animals and Gender

Sampling Time (Hours)

# MNPCEs per 10000 PCEs

Ratio PCEs / NCEs

M/5 24 11 1.04 F/5 24 14 1.07 M/5 48 9 0.92

PEG 400 0

F/5 48 22 1.27 M/5 24 15 0.99 F/5 24 12 1.34 M/5 48 5 1.23

100

F/5 48 17 1.08 M/5 24 10 0.90 F/5 24 13 1.31 M/5 48 20 0.95

400

F/5 48 28 1.16 M/5 24 8 1.24 F/5 24 9 1.48 M/5 48 15 0.95

TMC278

1600

F/5 48 15 1.00 40 mg/mL M/5 48 185 0.55 Cyclophosphamide

F/5 48 95 0.73 M: male; F: female, MNPCEs: micronucleated polychromatic erythrocytes; NCEs: normochromatic erythrocytes; PEG: polyethylene glycol, PCEs: polychromatic erythrocytes.

2.6.7.9.C. Genotoxicity: In Vivo Report Title: Mutagenicity Test on GS-4331-05 in the in vivo Mouse Micronucleus Assay


* Significantly greater than the corresponding vehicle control, p 0.01 Vehicle = Suspension vehicle. CP = Cyclophosphamide. PCE = Polychromatic erythrocyte. NCE = Normochromatic erythrocyte CONFIDENTIAL Page 217 17AUG2010

2.6.7.9.C. 97-TOX-4331-008: Mutagenicity Test on GS-4331-05 in the In Vivo Mouse Micronucleus Assay

Test for Induction of: Clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in PCE cells. (In vivo mouse micronucleus assay)

Treatment Schedule: Single dose at 500, 1000 & 2000 mg/kg

Study No: 97-TOX-4331-008

Species/Strain: Mouse, Crl: CD-1 (ICR) BR, Sampling Time: 28 & 48 hr post-dose Age: 9 weeks (approx) Method of Administration: Oral gavage GLP Compliance: Yes Cells Evaluated: Polychromatic erythrocyte (PCE) cells in bone marrow (target cells).

Vehicle Formulation: Suspension vehicle

Date of Dosing: 19

Number of Cells Analysed/Animal: 2000 PCEs Special Features: None Toxic/Cytotoxic Effects: TDF induced no signs of clinical toxicity in any of the treated animals and was not cytotoxic to the bone marrow Genotoxic Effects: TDF did not induce a statistically significant increase in micronuclei in bone marrow PCEs and is considered negative in the mouse bone marrow micronucleus test. Effects of the positive control: Cyclophosphamide induced statistically significant increases in micronucleated PCEs as compared to the vehicle controls, of 3.31 0.32%. Evidence of Exposure: TDF was detected in the plasma at all dose levels tested. Additional Information: Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCE) in at least the first 200 erythrocytes for each animal.

Treatment Dose

(mg/kg) Harvest Time (hr) % Micronucleated PCEs Mean of 2000 Per

Animal S.E. Males Ratio PCE:NCE Mean S.E.

Males Controls

Vehicle Vehicle 24 hr 0.02 0.02 0.35 0.05 48 hr 0.06 0.02 0.53 0.06

Positive CP 80.0 24 hr 3.31 0.32* 0.54 0.09 Test Article 500 24 hr 0.01 0.01 0.49 0.08

48 hr 0.02 0.01 0.51 0.06 1000 24 hr 0.03 0.02 0.38 0.03 48 hr 0.01 0.01 0.31 0.03 2000 24 hr 0.04 0.04 0.57 0.04 48 hr 0.04 0.02 0.41 0.06

2.6.7.9.D. Genotoxicity: In Vivo Report Title: In Vivo/In Vitro Unscheduled DNA Synthesis in Rat Primary Hepatocyte Cultures at 2 Timepoints



2.6.7.9.D. 23291-0-494-OECD: In Vivo/In Vitro Unscheduled DNA Synthesis in Rat Primary Hepatocyte Cultures at 2 Timepoints

Test for Induction of: DNA damage by measuring unscheduled DNA synthesis (UDS).

Treatment Schedule: TDF: Single dose at 0, 500, 1000, 2000 mg/kg. Postive Control: N-dimethylnitrosamine (DMN) at 10 & 15mg/kg

Study No: 23291-0-494-OECD

Species/Strain: Rat (Fischer 344) Sampling Time: TDF: 2-4 & 14-16 hours after dosing. Positive control: 2-4 hours after dosing for 10 mg/kg dose & 14-16 hours after dosing for 15 mg/kg dose

Age: 7–8 weeks (approx) Method of Administration: TDF: Oral gavage, Positive Control: Intraperitoneal injection

GLP Compliance: Yes

Cells Evaluated: Hepatocytes. Vehicle Formulation: 0.5% high viscosity carboxymethyl cellulose/0.8% benzyl alcohol/0.5% polysorbate 80

Date of Final Report: 20

Number of Cells Analysed/Animal: 150 cells/animal Special Features: None Toxic/Cytotoxic Effects: None - well tolerated to 2000 mg/kg Genotoxic Effects: Since one of two minimum criteria for a positive response was met at 2 time points, TDF was evaluated as weakly positive in the UDS assay. Evidence of Exposure: Additional Information: By all criteria DMN, the positive control, was considered positive in the UDS assay

2.6.7.9.D. Genotoxicity: In Vivo Report Title: In Vivo/In Vitro Unscheduled DNA Synthesis in Rat Primary Hepatocyte Cultures at 2 Timepoints



Test Article Dose (mg/kg) N1 Time (hr) Mean Nuclear Grains2 SD

Mean Net Nuclear

Grains3 SD

Mean Cytoplasmic Grains4 SD

% Cells with 5 NNG5

SD % Cells in S-Phase6

Vehicle 0 3 2–4 6.68 2.06 -0.53 0.69 7.21 1.60 3.33 3.16 0.47 Control 0 3 14–16 7.76 2.15 -0.19 0.60 7.96 1.70 5.78 5.04 0.40 DMN 10 3 2–4 35.99 7.24 27.25 5.78 8.74 1.86 97.78 2.91 0.60 15 3 14–16 19.07 6.11 12.38 4.95 6.69 1.40 78.83 20.66 0.42 TDF 500 3 2–4 6.89 1.50 0.90 1.18 5.98 0.95 11.56 8.93 0.49 3 14–16 6.68 1.35 1.32 1.02 5.36 0.59 10.89 7.29 0.27 1000 3 2–4 5.26 1.61 1.08 1.90 4.18 0.87 12.22 16.29 0.56 3 14–16 5.81 0.99 2.29 0.93 3.52 0.84 17.11 10.30 0.09 2000 3 2–4 6.29 1.70 1.88 1.03 4.41 0.87 17.78 10.27 0.20 3 14–16 7.71 0.91 2.65 0.76 5.06 0.57 23.66 8.38` 0.29

1 Number of animals analysed. 2 Average nuclear grain count 3 Average of net nuclear grain count with standard deviation (SD) between coverslips. Net nuclear grains (NNG) = Nuclear grain count – Average cytoplasmic grain count 4 Average of cytoplasmic grain count 5 Average percentage of cells with greater than or equal to 5 net nuclear grains 6 The percentage of heavily labeled cells observed Vehicle control article = Tenofovir DF suspension vehicle, 20 mL/kg. Positive control article = Dimethylnitrosamine (DMN), 1 mL/kg Criteria for a positive response: 2–4 hr timepoint – mean net nuclear grain counts 2.47 or nuclei containing 5 NNG 13.33 %. 14–16 hr time point – mean net nuclear

grain counts 2.81 or nuclei containing 5 NNG 15.78 %

2.6.7.10.A. Carcinogenicity Report Title: TP-0006: Two-Year Oral Oncogenicity Study in CD-1 Mice (TOX109)


2.6.7.10. Carcinogenicity 2.6.7.10.A. TOX109: TP-0006: Two-Year Oral Oncogenicity Study in CD-1 Mice

Species/ Strain: Mouse/ CD-1 (ICR) BR Duration of Dosing: 104 Weeks Study No: TOX109 Initial Age: 6 Weeks Method of Administration: Oral, gavage Date of First Dose: 20 Vehicle/Formulation: 0.5% Methylcellulose Testing Facility: Treatment of Controls: 0.5% Methylcellulose GLP Compliance: Yes Basis for High-Dose Selection: Results from 6 month mouse study; Toxicokinetic endpoint Special Features: None Daily Dose: (mg/kg) 0 (Control) 80 250 750 Gender M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Toxicokinetics From study AUC ( g hr/mL) (Week 26) nd* nd* 27.48 23.74 90.94 91.71 287.30 322.65 Number of Animals At Start 60 60 60 60 60 60 60 60 Died/Sacrificed Moribund 33 38 31 38 37 35 30 34 Terminal Sacrifice 27 22 29 22 23 25 30 26 Survival (%) 45 37 48 37 38 42 50 43 Body WeightA (%) 40.6 g 31.9 g +1 -1 0 -3 -3 -3 Food ConsumptionA (%) 6.0 g/day 5.8 g/day 0 +3 +3 +7 -2 +7

nd = not determined * Sample taken at Week 104, concentrations were generally below level of quantitation. A At 6 months; for controls group means are shown. For treated groups percent differences from control are shown.




Daily Dose: (mg/kg) 0 (Control) 80 250 750 Number Evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions

Adenoma, subcapsular cell, benign, primary

0 2 0 1 0 0 0 0 Adrenal Glands

Pheochromocytoma, benign, primary

0 0 0 0 0 1 0 0

Bone Osteoma, benign, primary 0 1 0 0 0 0 0 0 Bone Marrow, Femur Hemangiosarcoma, malignant,

primary 0 0 0 0 0 0 0 1

Bone, Vertebra Osteosarcoma, malignant, primary 0 0 0 0 0 0 0 1 Brain Astrocytoma, malignant, primary 0 0 1 0 0 0 0 0

Carcinoma, squamous cell, malignant, secondary

0 0 0 0 0 1 0 0

Fibrosarcoma, malignant, secondary

0 0 0 2 0 0 0 0

Hemangiosarcoma, malignant, primary

0 0 0 0 0 1 0 0

Leiomyosarcoma, malignant, primary

0 0 0 0 0 1 0 0

Liposarcoma, malignant, secondary

0 1 0 0 0 0 0 0

Cavity, Abdominal

Osteosarcoma, malignant, primary 0 1 0 0 0 0 0 0 Cavity, Oral Carcinoma, squamous cell,

malignant, primary 1 0 0 0 0 0 0 0

Fibrosarcoma, malignant, primary 0 0 0 1 0 0 0 0 Fibrosarcoma, malignant, secondary

0 0 0 1 0 0 0 0 Cavity, Thoracic


0 1 0 0 0 0 0 0

Gall Bladder Adenoma, benign, primary 0 0 1 0 0 0 1 1 Adenocarcinoma, malignant, primary

1 2 1 0 0 1 0 1 Harderian Glands

Adenoma, benign, primary 4 1 4 3 6 5 4 5 Heart Hemangiosarcoma, malignant,

primary 0 0 0 0 0 1 0 0

Kidneys Adenoma, tubular cell, benign, primary

1 0 0 0 2 0 0 0





Adenoma, hepatocellular, benign, primary

5 0 7 0 7 1 4 2

Carcinoma, hepatocellular, malignant, primary

3 1 4 0 0 1 1 0


0 0 0 1 0 0 0 0

Liver


2 0 1 0 2 2 2 0

Adenocarcinoma, malignant, secondary

0 0 0 1 0 0 0

Adenoma, bronchiolar alveolar, benign, primary

7 9 14 15 15 8 13 6

Carcinoma, bronchiolar alveolar, malignant, primary

1 1 2 3 0 1 1 1

Lung


0 0 0 2 0 1 0 0

Lymph Node, Axillary Fibrosarcoma, malignant, secondary

0 0 0 1 0 0 0 0

Lymph Node, Cervical Lymphangioma, benign, primary 0 0 0 0 0 0 0 1 Lymph Node, Hepatic Leiomyosarcoma, malignant,

secondary 0 0 0 0 0 1 0 0


0 0 0 1 0 0 0 0 Lymph Node, Mediastinal


0 1 0 0 0 0 0 0

Lymph Node, Mesenteric Leiomyosarcoma, malignant, secondary

0 0 0 0 0 1 0 0

Lymph Node, Renal Leiomyosarcoma, malignant, secondary

0 0 0 0 0 1 0 0

Mammary Gland Adenocarcinoma, malignant, primary

0 0 0 1 0 0 0 1

Mesentery/Peritoneum Leiomyoma, benign, primary 0 1 0 0 0 0 0 0 Multicentric Neoplasm Leukemia, granulocytic,

malignant, muticentric 0 1 0 0 0 0 0 0





Lymphoma, malignant, multicentric

4 5 0 7 3 6 4 4

Sarcoma, histiocytic, malignant, multicentric

0 6 0 2 1 6 1 2

Cystadenoma, benign, primary NA 0 NA 1 NA 2 NA 1 Hemangioma, benign, primary NA 1 NA 0 NA 0 NA 0 Leiomyosarcoma, malignant, secondary

NA 0 NA 0 NA 1 NA 0

Sex-cord/stromal tumor, benign, primary B

NA 0 NA 0 NA 0 NA 2

Ovaries

Sex-cord/stromal tumor, malignant, primary

NA 1 NA 0 NA 1 NA 0


0 0 0 1 0 0 0 0

Leiomyosarcoma, malignant, secondary

0 0 0 0 0 1 0 0

Pancreas


0 1 0 0 0 0 0 0

Pituitary Gland Adenoma, pars distalis, benign, primary

0 1 0 2 0 0 0 0

Skeletal Muscle Leiomyosarcoma, malignant, secondary

0 0 0 0 0 1 0 0

Skin Carcinoma, squamous cell, malignant, primary

0 0 0 0 0 1 1 0

Fibrosarcoma, malignant, primary 1 1 0 2 1 5 1 1 Skin, Subcutis Liposarcoma, malignant, primary 0 1 0 0 0 0 0 0

Small Intestine, Ileum Adenocarcinoma, malignant, primary

0 1 0 0 0 0 0 0


0 0 0 1 0 0 0 0 Spleen

Hemangiosarcoma, malignant, primaryC

0 0 1 1 2 0 3 1

NA = not applicable B Peto Test; P-value 0.040 C Peto Test; P-value 0.031




Daily Dose: (mg/kg) 0 (Control) 80 250 750 Number Evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions Testes Adenoma, interstitial cell, benign,

primary 0 NA 0 NA 2 NA 1 NA

Thymus Gland Fibrosarcoma, malignant, secondary 0 0 0 1 0 0 0 0 Ademoma, c-cell, benign, primary 0 1 0 0 0 0 0 0 Thyroid Gland Adenoma, follicular cell, benign, primary

0 1 0 0 0 0 0 0

Tooth/Teeth Odontoma, benign, primary 0 0 1 0 0 0 0 0 Leiomyosarcoma, malignant, secondary

0 0 0 0 0 1 0 0 Urinary Bladder

Mesenchymal tumor, benign, primary

0 1 0 0 0 0 0 0

Adenocarcinoma, malignant, primary

NA 0 NA 3 NA 1 NA 0

Carcinoma, squamous cell, malignant, primary

NA 0 NA 1 NA 0 NA 0


NA 0 NA 1 NA 0 NA 1

Leiomyoma, benign, primary NA 4 NA 4 NA 1 NA 2 Leiomyosarcoma, malignant, primary

NA 0 NA 0 NA 0 NA 1

Leiomyosarcoma, malignant, secondary

NA 0 NA 0 NA 1 NA 0

Polyp, glandular, benign, primary NA 1 NA 1 NA 0 NA 0 Polyp, stromal, benign, primary NA 7 NA 5 NA 2 NA 3

Uterus with Cervix

Sarcoma, stromal, malignant, primary

NA 0 NA 3 NA 1 NA 1

Vagina Polyp, stromal, benign, primary NA 1 NA 0 NA 0 NA 0

NA = not applicable Noteworthy Findings:

Gross Pathology: None Microscopic Pathology – Non-neoplastic lesions: None

2.6.7.10.B. Carcinogenicity Report Title: TP-0006: Two-Year Oral Oncogenicity Study in CD Rats (TOX108)



2.6.7.10.B. TOX108: TP-0006: Two-Year Oral Oncogenicity Study in CD Rats

Species/ Strain: Rat/ CD [Crl:CD (SD) IGS BR] Duration of Dosing: 104 Weeks Study No: TOX108 Initial Age: 6 Weeks Method of Administration: Oral, gavage Date of First Dose: 20 Vehicle/Formulation: 0.5% Methylcellulose Testing Facility: Treatment of Controls: 0.5% Methylcellulose GLP Compliance: Yes Basis for High-Dose Selection: Results from 90 day rat study; toxicokinetic endpoint Special Features: None Daily Dose: (mg/kg) 0 (Control) 60 200 600 Gender M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Toxicokinetics From study AUC0-24 ( g.h/mL) (Week 26) nd* nd* 42.87 52.53 137.42 170.68 326.77 404.07 Number of animals At Start 60 60 60 60 60 60 60 60 Died/Sacrificed Moribund 31 36 40 28 34 31 32 40 Terminal Sacrifice 29 24 20 32 26 29 28 20 Survival (%) 48 40 33 53 43 48 47 33 Body Weight A (%) 584 g 339 g 0 -4 +2 0 +3 +1 Food Consumption A (%) 26 g/day 20 g/day +4 0 +8 +5 +8 +10 nd = not determined * Sample taken at Week 104, concentrations were below level of quantitation. A At 6 months; for controls group means are shown. For treated groups percent differences from control are shown.





Hibernoma, benign, primary

1 0 1 0 0 0 0 0 Adipose Tissue, Brown

Hibernoma, malignant, primary

0 1 0 0 0 1 0 1

Adenoma, cortical, benign, primary

0 1 0 1 1 0 1 1

Carcinoma, cortical, malignant, primary

0 3 1 1 0 0 0 0

Pheochromocytoma, benign, primary

4 1 7 1 12 1 7 2

Adrenal Glands

Pheochromocytoma, malignant, primary

0 0 1 0 0 1 0 1

Bone, Tibia Hemangiosarcoma, malignant, secondary

0 0 1 0 0 0 0 0

Astrocytoma, malignant, primary

2 0 0 0 1 1 0 0

Carcinoma, pars distalis, malignant, secondary

0 0 0 0 1 0 0 0

Carcinoma, zymbals gland, malignant, secondary

0 0 0 0 1 1 0 0

Granular cell tumor, benign, primary

0 1 1 0 0 1 0 0

Brain

Reticulosis, malignant, primary

0 1 0 0 0 0 0 0

Carcinoma, malignant, primary

0 0 0 1 0 0 0 0

Fibrosarcoma, malignant, primary

1 0 0 0 0 0 0 0

Cavity, Abdominal

Lipoma, benign, primary

0 0 0 0 0 0 1 1

Neuroendocrine tumor, benign, primary

1 0 0 0 0 0 0 0





Hibernoma, benign, primary 0 1 0 0 0 0 0 0 Cavity, Thoracic Osteosarcoma, malignant, primary

0 0 0 0 1 0 0 0

Coagulating Glands

Adenoma, benign, primary 0 0 0 0 0 0 1 0

Ears Fibroma, benign, primary

1 0 0 0 0 0 0 0

Hard Palate Fibrosarcoma, malignant, primary

0 0 1 0 0 0 0 0

Harderian Glands Carcinoma, zymbals gland, malignant, secondary

1 0 0 0 0 0 0 0

Implantation Site Fibrosarcoma, malignant, primary

0 0 0 0 0 0 1 0

Heart Adenocarcinoma, malignant, secondary

0 1 0 0 0 0 0 0

Carcinoma, tubular cell, malignant, primary

0 0 1 0 0 1 0 0


0 1 0 0 0 0 0 0

Kidneys

Liposarcoma, malignant, primary

1 1 0 0 0 1 0

Large Intestine, Cecum


1 0 0 0 0 0 0 0

Large Intestine, Rectum

Granular cell tumor, malignant, secondary

0 0 0 1 0 1 0 0





Adenoma, hepatocellular, benign, primary

1 3 0 0 0 1 1 2 Liver

Carcinoma, hepatocellular, malignant, primary

0 0 0 1 0 0 0 0

Adenoma, bronchiolar alveolar, benign, primary

1 0 0 0 0 0 0 1

Hibernoma, malignant, secondary

0 1 0 0 0 0 0 0

Lung


1 0 0 0 0 0 0 0

Lymph Node, Cervical

Carcinoma, c-cell, malignant, secondary

0 0 0 0 0 0 1 0

Lymph Node, Mediastinal

Adenocarcinoma, malignant, secondary

0 1 0 0 0 0 0 0

Lymph Node, Mesenteric


0 0 0 1 0 0 0 0


0 18 1 9 1 13 0 7

Adenoma, benign, primary

0 3 0 2 0 3 0 1

Mammary Gland

Fibroadenoma, benign, primary

0 22 2 17 1 18 0 16

Lymphoma, malignant, multicentric

1 1 2 0 1 2 1 0 Multicentric Neoplasm

Sarcoma, histiocytic, malignant, multicentric

0 1 2 0 0 1 1 2

Nose, Level B Carcinoma, zymbals gland, malignant, secondary

1 0 0 0 0 1 0 0




Daily Dose: (mg/kg) 0 (Control) 60 200 600 Number Evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions Lymphangioma, benign,

primary 0 0 0 0 1 0 0 0


1 0 0 0 0 1 0 0 Nose, Level C

Papilloma, benign, primary

0 0 0 0 1 0 0 0


1 0 0 0 0 1 0 0 Nose, Level D

Polyp, benign, primary

0 0 0 0 1 0 0 0

Ovaries Sex-cord/stromal tumor, benign, primary

NA 1 NA 0 NA 1 NA 0

Adenoma, acinar cell, benign, primary

0 0 0 0 0 0 1 0

Adenoma, islet cell, benign, primary

2 3 3 0 3 0 3 2

Carcinoma, islet cell, malignant, primary

0 0 1 0 1 0 1 0

Carcinoma, malignant, secondary

0 0 0 1 0 0 0 0

Fibroma, benign, primary

0 0 0 0 0 0 0 1

Pancreas


1 0 0 0 0 0 0 0

Parathyroid Glands

Adenoma, benign, primary 0 0 0 0 4 1 0 0

Carcinoma, pars distalis, malignant, primary

0 0 0 0 1 1 0 1

NA = not applicable






0 NA 0 NA 1 NA 0 NA Seminal Vesicles


1 NA 0 NA 0 NA 0 NA


1 0 1 0 0 0 0 1

Hemangioma, benign, primary

0 0 0 0 1 0 0 0

Papilloma, squamous cell, benign, primary

0 0 2 0 1 0 0 0

Skin

Trichoepithelioma, benign, primary

0 0 1 0 1 0 0 0

Fibroma, benign, primary

0 1 2 2 4 0 2 0

Fibrosarcoma, malignant, primary

1 1 0 0 0 0 0 1


0 0 1 0 0 0 0 0

Hibernoma, benign, primary

0 1 0 0 0 0 0 0


0 1 0 1 0 0 1 0

Skin, Subcutis

Schwannoma, malignant, primary

1 0 0 0 1 0 0 0

Small Intestine, Duodenum

Carcinoma, malignant, secondary

0 0 0 1 0 0 0 0

Spleen Carcinoma, malignant, secondary

0 0 0 1 0 0 0 0

Stomach, Glandular


1 0 0 0 0 0 0 0





Osteosarcoma, malignant, primary

0 0 1 0 0 0 0 0

Testes Adenoma, interstitial cell, benign, primary

0 NA 0 NA 1 NA 1 NA

Lymphoma, malignant, primary

0 0 0 1 0 0 0 0

Thymoma, benign, primary

0 0 1 0 0 0 0 0

Thymus gland

Thymoma, malignant, primary

1 0 0 0 1 0 0 0

Ademoma, c-cell, benign, primary

3 5 2 2 7 3 0 1

Adenoma, follicular cell, benign, primary

7 1 0 0 4 0 1 3

Carcinoma, follicular cell, malignant, primary

0 1 0 0 0 0 0 0

Thyroid Gland

Carcinoma, c-cell, malignant, primary

1 0 1 0 0 0 1 0

Tongue Carcinoma, squamous cell, malignant, primary

0 0 0 1 0 0 0 0


0 0 1 0 0 0 0 0

Carcinoma, squamous cell, malignant, secondary

0 0 0 0 1 0 0 0

Urinary Bladder

Carcinoma, transitional cell, malignant, primary

0 0 0 0 1 0 0 0


NA 0 NA 1 NA 0 NA 1 Uterus with Cervix


NA 6 NA 6 NA 4 NA 2





Granular cell tumor, benign, secondary

NA 1 NA 0 NA 0 NA 0

Granular cell tumor, malignant, secondary

NA 0 NA 0 NA 1 NA 0

Leiomyoma, benign, primary

NA 0 NA 0 NA 1 NA 0

Polyp, stromal, benign, primary

NA 6 NA 3 NA 4 NA 6

Sarcoma, stromal, malignant, primary

NA 0 NA 0 NA 1 NA 1

Schwannoma, malignant, primary

NA 0 NA 0 NA 0 NA 1


NA 1 NA 2 NA 4 NA 1

Granular cell tumor, malignant, primary

NA 0 NA 0 NA 1 NA 0

Polyp, stromal, benign, secondary

NA 0 NA 0 NA 1 NA 0

Vagina

Sarcoma, stromal, malignant, secondary

NA 0 NA 0 NA 0 NA 1

Zymbal’s Gland Carcinoma, zymbals gland, malignant, primary

1 0 1 0 1 1 0 0

NA = not applicable Noteworthy Findings:

Gross Pathology: None Microscopic Pathology – Non-neoplastic lesions: None

2.6.7.10.C. Carcinogenicity Report Title: Carcinogenicity Study by Oral Gavage Administration to CD-1 Mice for 104 Week



2.6.7.10.C. TMC278-NC120: Carcinogenicity Study by Oral Gavage Administration to CD-1 Mice for 104 Weeks

Species/strain: Mouse/Crl:CD-1™ (ICR) Age at first dose: 41 to 48 days Duration of dosing: 104 weeks Duration of postdose: none

Administration route/method: Oral/gavage Doses: 0, 20, 60, 160 mg base eq./kg/day TMC278.HCl Test article batch:

Vehicle/formulation: 0.5% w/v Methocel/Suspension

Testing facility: Study no.: TMC278-NC120 GLP compliance: Yes

No Observed Adverse Effect Level: Not established Toxicokinetics Dose (mg eq./kg/day) 0 (vehicle) a 20 60 160 No. of animals M:18 F:18 M:24 F:24 M:24 F:24 M:24 F:24 AUC ( g.h/mL) Day 1 (AUC0- ) / / 59 61 239 182 440 345 Week 28 (AUC0-24h) / / 76 51 230 278 505 766 a TMC278 plasma concentrations above the lower limit of quantification (0.002 g/mL) were found in 10 out of 42 samples (ranging between 0.002 and 0.012 g/mL) Noteworthy findings Dose (mg eq./kg/day) 0 (vehicle) 20 60 160 No. of animals M:60 F:60 M:60 F:60 M:60 F:60 M:60 F:60 Died or Sacrified Moribund 25 40 35 37 39 30 42 43

Body Weightb 48.4 37.8 1.02 1.05 1.03 1.09 1.01 1.05 Body Weight Gainb 15.4 13.6 1.06 1.13 1.10 1.23* 1.07 1.17* Food Consumptionb 35 35 1.06 1.00 1.09 1.06 1.23 1.14 Clinical Observations - - - - - - - - Hematology - - - - - - - - Organ Weights Liver (absolute) 2.334 - 1.35** - 1.69** - 1.18** - Liver (adjusted) - 1.999 - 1.13 - 1.39** - 1.94**




Study: TMC278-NC120: Carcinogenicity Study by Oral Gavage Administration to CD-1 Mice for 104 Weeks (continued) Noteworthy findings Dose (mg eq./kg/day) 0 (vehicle) 20 60 160 No. of animals M:60 F:60 M:60 F:60 M:60 F:60 M:60 F:60 Gross Pathology Liver: Enlarged 2 7 3 7 2 10 11 30 Masses 13 4 21 9 23 13 29 32 Pale area(s) 1 6 4 9 6 10 12 33 Lungs: Mass(es) 23 - 21 - 26 - 30 - Kidney: Left kidney Granular - 6 - 6 - 4 - 15 Right Kidney Granular - 6 - 6 - 4 - 14 b At end of dosing period. For vehicle, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data (not

on the multiples of control/baseline). - No noteworthy findings, * - p<0.05 ** - p<0.01; F: female; M: male




Dose (mg eq./kg/day) 0 (vehicle) 20 60 160 No. of animals M:60 F:60 M:60 F:60 M:60 F:60 M:60 F:60 Histopathologya Liver (n°. examined) 60 60 60 60 60 60 60 60 - M-Hepatocellular carcinoma 4 0 7 0 7 2 9 7 - B-Hepatocellular adenoma 8 1 14 0 21 8 19 16 - Eosinophilic foci 0 0 0 0 1 1 3 8 - Basophilic foci 0 0 4 0 0 2 5 10 - Clear cell foci 0 0 3 1 1 1 4 4 - Generalised eosinophilic hepatocyte hypertrophy

0 1 1 0 1 11 5 42

- Brown pigmented macrophages associated with mononuclear cells

0 2 0 3 1 7 3 17

- Brown pigmented Kupffer cells/macrophages

0 5 3 3 2 17 9 17

Lungs (n°. examined) 60 60 60 60 60 60 60 60 - M-Bronchioloaveolar adenocarcinomas

6 - 5 - 0 - 10 -

Kidney (n°. examined) - 60 - 60 - 60 - 60 Kidney (Left) - Glomerulonephritis - 7 - 7 - 6 - 11 - Nephropathy 27 21 23 37 Kidney (Right) - Glomerulonephritis - 7 - 7 - 6 - 11 - Nephropathy - 22 - 20 - 20 - 36 - No noteworthy findings; F: female; M: male; a Histopathology findings: M = malignant; B = benign

2.6.7.10.D. Carcinogenicity Report Title: Carcinogenicity Study by Oral Gavage Administration to CD-1 Mice for 104 Week



2.6.7.10.D. TMC278-NC123: Carcinogenicity Study by Oral Gavage Administration to CD Rats for 104 Weeks

Species/strain: Rat/Crl: CD/Sprague Dawley Age at first dose: approximately 42 days Date of first dose: 20 Duration of dosing: 104 Weeks (males), 98 weeks (Group 1, 3, 4 and 5 females), 97 weeks ( Group 2 females) Duration of postdose: None

Administration route/method: Oral/Gavage Doses: 0, 40, 200, 500, 1500 mg base eq./kg/day TMC278.HCl Test article batch:

Vehicle/formulation: 0.5% w/v Methocel in purified water/suspension

Testing facility: Study No.: TMC278-NC123 GLP compliance: Yes

No Observed Adverse Effect Level: / Toxicokinetics: Dose (mg base eq./kg/day) 0 (vehicle) b 40 200 500 1500 No. of Animals M:6 F:6 M:9 F:9 M:9 F:9 M:9 F:9 M:9 F:9 AUC ( g.h/mL) a Day 1 / / 19 17 34 40 c 45 63 58 81 Week 27 / / 4.5 15 11 36 14 70 20 81 Week 39 / / 6.3 14 8.2 41 14 46 18 84 a AUC0- on Day 1 otherwise AUC0-24h; b on Day 1, 2 samples out of 18 were above the limit of quantification (LOQ, 0.001-0.002 g/mL), at Week 27, 17 samples out of 18 were

above LOQ and at Week 39, 7 samples out of 18 were above the LOQ; c AUC0-24h. Noteworthy Findings: Dose (mg base eq./kg/day) 0 (vehicle) 40 200 500 1500 No. of Animals M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 Died or Sacrificed Moribund 40 45 40* 46 38 35 37 33 44 34

Body Weight - - - - - - - - - - Body Weight Gain - - - - - - - - - - Food consumption - - - - - - - - - - Ophthalmoscopy - - - - - - - - - - * includes one animal that was found dead during the necropsy period (Week 105), - No noteworthy findings; F: female; M: male




Study: TMC278-NC123: Carcinogenicity Study by Oral Gavage Administration to CD Rats for 104 Weeks (continued) Dose (mg base eq./kg/day) 0 (vehicle) 40 200 500 1500 No. of Animals M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 Clinical Observations - Encrustations, head - 9 - 13 - 14 - 15 - 19 - Brown staining, head - 19 - 13 - 14 - 22 - 31 - Brown staining, dorsal body surface

7 - 9 - 6 - 10 - 15 -

- Yellow staining, ventral body surface

4 - 4 - 1 - 2 - 14 -

- Hairloss, forelimbs 1 6 6 9 2 10 4 9 10 12 - Hairloss, head - 22 - 22 - 20 - 24 - 33 - Ungroomed 12 - 17 - 21 - 20 - 23 - Palpable swellings - - - - - - - - - - Hematology - - - - - - - - - - Serum Chemistry – Week 52 of treatmenta

- Alkaline phosphatase U/L 53 24 1.06 1.04 1.13 1.21 1.42** 1.08 1.92** 1.29* - Alanine aminotransferase U/L 43 - 1.02 - 1.02 - 1.49** - 2.63** - - Aspartate aminotransferase U/L

74 - 1.16 - 1.09 - 1.50* - 1.92** -

- gamma glutamyltransferaseGT U/L

1 - 1.00 - 2.00 - 2.00* - 2.00* -

- Urea mmol/L 4.10 - 0.94 - 1.01 - 1.08 - 1.14** - - Creatinine μmol/L 41 - 1.02 - 1.12* - 1.22** - 1.20** - - Cholesterol mmol/L 2.45 2.99 1.00 0.89 0.91 0.86* 0.84 0.80** 0.91 0.77** - Triglycerides mmol/L 1.71 2.12 0.84 0.98 0.73* 0.78 0.80* 0.57** 0.76* 0.57** - Chloride mmol/L 99 - 1.00 - 1.00 - 1.00 - 0.98** - - Calcium mmol/L 2.69 2.80 0.99 0.99* 0.99* 0.97** 0.98** 0.97** 0.97** 0.96** - Albumin g/L 31 - 1.00 - 1.03 - 1.06** - 1.10** - - a1 g/L - 14 - 0.93* - 0.86** - 0.86** - 0.93** - a2 g/L 4 - 0.75** - 0.75** - 0.75** - 0.50** - - Beta g/L 17 15 0.94 1.00 0.94* 0.93* 0.88** 0.93** 0.88** 0.93** - A/G ratio 0.74 1.13 1.05 1.01 1.08* 1.07 1.19** 1.13** 1.18** 1.06**

a At end of dosing period. For vehicle, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data (not on the multiples of control/baseline), - No noteworthy findings, * - p<0.05, ** - p<0.01, *** - p<0.001; F: female; M: male




Study: TMC278-NC123: Carcinogenicity Study by Oral Gavage Administration to CD Rats for 104 Weeks (continued) Dose (mg base eq./kg/day) 0 (vehicle) 40 200 500 1500 No. of Animals M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 Serum Chemistry - TERMa - Alkaline phosphatase U/L 58 - 1.10 - 1.41* - 1.81** - 2.19** - - Creatinine mol/L 33 40 1.06 1.18** 1.21# 0.98 1.21** 1.15* 1.15** 1.33** - Glucose mmol/L 5.72 - 1.11 1.15 1.11 1.19** - Cholesterol mmol/L 3.42 - 0.98 - 0.88* - 0.82* - 0.85* - - Triglycerides mmol/L - 1.27 - 1.23 - 0.86 - 0.83 - 0.72 - Potassium mmol/L - 3.7 - 1.08 - 1.08 - 1.11* - 1.19** - Sodium mmol/L 143 - 1.01 - 1.01 - 1.00 - 0.99* - - Chloride mmol/L 100 - 1.00 - 1.02** - 1.02** - 1.02** - - Calcium mmol/L - 2.83 - 0.98* - 0.98 - 0.98* - 0.98* - Phosphorus mmol/L - 1.41 - 1.06 - 0.96 - 0.99 - 1.15** - Albumin g/L 30 - 1.00 - 0.97 - 1.03 - 1.07** - - Beta g/L 21 - 0.90* - 0.90* - 0.86** - 0.81** - - A/G ratio 0.64 - 1.06 - 1.02 - 1.11* - 1.22** - Urinalysis - Week 51 of treatmenta

- pH - 6.9 - 0.94 - 0.96 - 1.01 - 0.87** - Protein g/L - 0.27 - 0.59 - 0.89 - 0.56 - 1.11** - Potassium mmol/L - 160.4 - 0.94 - 0.86 - 0.86 - 0.81* - Chloride mmol/L 45.1 31.6 1.10 1.13 1.14 1.59** 1.80** 2.75** 3.63** 4.80** Urinalysis – TERM - pH 7.9 6.6 1.03 0.97 1.03 1.00 1.00 1.03 0.90** 0.91** - Potassium mmol/L 130.7 107.2 0.98 1.06 0.81* 0.97 0.80* 0.82** 0.85* 0.85** - Chloride mmol/L 42.2 34.9 0.95 1.01 1.29 1.47** 1.47** 1.94** 3.48** 3.46** Organ Weights, Unadjusted (absolute)

Thyroid and Parathyroid 0.033 0.027 - - - - - - 1.64* 1.37** a At end of dosing period. For vehicle, group means are shown. For treated groups, multiples of control/baseline are shown. Statistical significance is based on actual data (not

on the multiples of control/baseline), TERM: End of treatment period, - : No noteworthy findings, # : With exclusion of 3M, value 148 mol/L, *: p<0.05, ** : p<0.01, *** : p<0.001; F: female; M: male




Study: TMC278-NC123: Carcinogenicity Study by Oral Gavage Administration to CD Rats for 104 Weeks (continued) Dose (mg base eq./kg/day) 0 (vehicle) 40 200 500 1500 No. of Animals M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 Gross Pathology Number Examined: 65 65 65 65 65 65 65 65 65 65 - Lungs and Bronchi, pale area(s)

22 17 37 25 31 23 31 28 40 42

- Liver, dark area(s) 12 - 12 - 20 - 11 - 17 - - Liver, mass(es) - 5 - 11 - 8 - 10 - 7 - Thyroids, mass(es) 2 - 6 - 6 - 3 - 4 - - Thyroids, enlarged - 2 - 3 - 3 - 4 - 12 - Abnormal contents (pale/ white) of GI Tract

6 4 9 6 18 14 15 13 32 19

Histopathology Liver (n°. examined) 65 65 65 65 65 65 65 65 65 65 - Hepatocelluar adenoma 0 1 1 5 3 5 3 6 3 4 - Sinuosoidal dilatation:

Minimal 0 11 - 6 - 13 - 13 - 12 Slight 0 8 - 22 - 19 - 14 - 28 Moderate 0 6 - 2 - 4 - 4 - 3 Total 0 25 - 30 - 36 - 31 - 43

Thyroid (n°. examined) - Follicular cell adenoma 1 0 3 0 4 3 5 3 5 4 - Follicular cell carcinoma 0 0 0 1 2 1 1 0 2 1 - Follicular cell hypertrophy:

Minimal 1 0 0 1 2 3 1 1 1 6 Slight 0 0 1 1 2 1 3 1 3 2 Total 1 0 1 2 4 4 4 2 4 8

- Cystic follicular cell hyperplasia:

Minimal 3 0 1 1 3 2 0 2 5 2 Slight 3 0 4 1 2 0 2 3 1 3 Moderate 0 0 0 0 0 0 0 0 1 0 Total 6 0 5 2 5 2 2 5 7 5

- : No noteworthy findings ; F: female; M: male




Study: TMC278-NC123: Carcinogenicity Study by Oral Gavage Administration to CD Rats for 104 Weeks (continued) Dose (mg base eq./kg/day) 0 (vehicle) 40 200 500 1500 No. of Animals M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 Histopathology (cont’d) Nasal turbinates (n°. examined)

65 65 65 65 65 65 65 65 65 65

- Inflammatory exudate 1 0 2 0 2 0 3 1 39 26 - Respiratory epithelium hyperplasia:


- Respiratory epithelium inflammation:


- Olfactory epithelium respiratory metaplasia:


Lungs (n°. examined) 65 65 65 65 65 65 65 65 65 65 - Foamy alveolar macrophages:

Minimal 31 31 32 32 33 38 30 36 24 35 Slight 11 4 18 3 11 3 16 9 19 12 Moderate 2 0 1 2 2 1 1 0 2 6 Marked 0 0 0 0 1 0 0 0 0 0 Total 44 35 51 37 47 42 47 45 45 53

F: female; M: male




Study: TMC278-NC123: Carcinogenicity Study by Oral Gavage Administration to CD Rats for 104 Weeks (continued) Dose (mg base eq./kg/day) 0 (vehicle) 40 200 500 1500 No. of Animals M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 M:65 F:65 Histopathology (cont’d) Lungs (n°. examined) 65 65 65 65 65 65 65 65 65 65 - Alveolar epithelial hyperplasia:


F: female; M: male

2.6.7.10.E. Carcinogenicity Report Title: An Oral Carcinogenicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Mouse



2.6.7.10.E. M990205: Carcinogenicity: An Oral Carcinogenicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Mouse

Species/ Strain: Mouse, Swiss Crl:CDR-1(ICR)BR Duration of Dosing: 104 weeks Study No: M990205 Initial Age: 6 Weeks (approx) Method of Administration: Oral gavage Date of First Dose: 20 (Main) Vehicle/ Formulation: Suspension vehicle Treatment of Controls: Oral gavage (vehicle) GLP Compliance: Yes Basis for High-Dose Selection: The dose levels were selected according to the potential human exposure, existing toxicity data and limitations imposed by the test article. A previous 13 week oral toxicity study indicated that 600 mg/kg/day (60 mg/mL) is the highest dose level of Tenofovir DF that may be practically administered to albino mice. Special Features: Separate populations for toxicokinetic evaluations were included in the study: Groups of 30 males and 30 females were sampled on Days 1 and 180 for the high dose only. Animals were euthanized on completion of the blood collection & subjected to necropsy. Before assignment of animals to a treatment group, 10 males & 10 females animals, selected randomly, were subjected to a health screen. Daily Dose: (mg/kg) 0 (Control) 100 300 600

M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Toxicokinetics AUCss 0-24 ( g h/mL) (Day 1) - - - - - - 30.9 35.1 AUCss 0-24 ( g h/mL) (Day 180) - - - - - - 44.3 50.9 Number of Animals At Start 60 60 60 60 60 60 60 60 Died/Sacrificed Moribund 33 39 37 32 31 29 40 43 Terminal Sacrifice 27 21 23 28 29 31 20 17 Survival (%) 45 35 38 47 48 52 33 28 Body Weight (%) A 38.65g 30.79g -4.4* +1.5% -3.4 -1.6% -3.7% -0.07% Food Consumption (%) A 37.4g 39.4g +8.6% +2.2% +5.1% +0.3% +9.9% -0.3%

Dunnetts “t” test * p 0.05 A At 6 months, for controls group means are shown. For treated groups percent differences from controls are shown. Statistical differences are based upon the actual data not

percent differences.





Leiomyosarcoma - - 1 - - - - 1 Osteosarcoma - - - 1 - - - - Rhabdomyosarcoma (metastasis)

- - 1 - - - - -

Abdomen

Sarcoma - - - 1 - - - - Adenoma: cortical - 1 - 1 1 1 - - Sarcoma: fusiform cell 1 - 1 - - - - Benign pheochromocytoma - - - 2 - - - -

Adrenal

Sarcoma (metastasis) - - - 1 - - - - Bone - Femur Hemangiosarcoma - - 1 - - - - -

Hemangiosarcoma 1 - - - - - - - Osteosarcoma - - - 1 - - - -

Bone - Misc

Rhabdomyosarcoma - - - - - 1 - - Brain Malignant meningioma 1 - - - - - - - Cecum Leiomyoma - - - - - 1 - -

Rhabdomyosarcoma (metastasis)

- - 1 - - - - - Diaphragm

Sarcoma(metastasis) - - - 1 - - - - Adenoma - - - - - - - 1 Duodenum Adenocarcinoma - - - - - - 1 1

Epididmydis Rhabdomyosarcoma (metastasis)

- - 1 - - - - -

Meningioma 1 - - - - - - - Eye Carcinoma (metastasis) 1 - - - - - - -




Daily Dose: (mg/kg) 0 (Control) 100 300 600 Number evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions Fat Leiomysarcoma (metastasis) - 1 - - - - - - Harderian Gland Adenoma 1 2 5 - 3 3 7 3 Head Hemangiosarcoma - - 1 - - - - -

Malignant lymphoma 3 10 1 17 3 4 2 5 Hemolym Tissue Histiocytic sarcoma - 9 1 4 - 5 1 1

Adenoma - - - - 1 - - - Jejunum Adenocarcinoma - - - 1 1 - - 1 Carcinoma: tubular cell - - 1 - - - - - Kidney Sarcoma (metastasia) - - - 1 - - - - Adenoma: hepatocellular 5 1 1 1 2 - 4 9 Carcinoma: hepatocellular 6 - 2 - - - - - Hemangioma 1 - - - - - 2 - Hemangiosarcoma 2 - 1 - - 2 - 1 Leiomyosarcoma (metastasis) - 1 1 - - - - - Rhabdomyosarcoma (metastasis)

- - 1 - - - - -

Liver

Carcinoma: metastasis 1 - - - - - - - Carcinoma: metastasis 1 1 - - - 1 - - Lung Adenoma: alveolar/bronchiolar

10 10 8 9 11 5 10 7




Daily Dose: (mg/kg) 0 (Control) 100 300 600 Number evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions

Adenocarcinoma: Alveolar/bronchiolar

9 5 7 2 9 2 9 1

Mammary carcinoma (metastasis)

- 1 - - - - - -


- - 1 - - - - -

Sarcoma (metastasis) - - - 1 - - - - Carcinosarcoma (metastasis)

1 - - - - - - 1

Lung Cont’d

Fibroma - - - - - 1 - - Hemangioma 1 - - - - - - - Carcinoma (metastasis) 1 - - - - - - -

Lymph Node

Mammary Carcinoma (metastasis)

- 1 - - - - - -

Hemangioma 1 1 - - - - - - Lymph Node Mesenteric Rhabdomyosarcoma - - 1 - - - - -

Adenocarcinoma - 3 - - - - - - Mammary Gland Adenoacanthoma - 1 - - - 1 - -

Muscle Skeletal Hemangioma - 1 - - - - - - Rhabdomyosarcoma - - 1 - - - - - Muscle Skeletal

Misc. Sarcoma (Metastasis) - - - 1 - - - -





Cystadenoma - 1 - 1 - - - - Carcinoma: tubulostromal - - - 1 - - - - Malignant granulosa-theca cell tumor

- 1 - - - - - -

Leiomyosarcoma (metastasis)

- 1 - - - - - -

Ovary

Carcinoma (metastasis) - - - - - 1 - - Adenocarcinoma (metastasis)

- - - - 1 - - -

Carcinoma (metastasis) - - - - - 1 - - Leiomyosarcoma (metastasis)

- 1 - - - - - -

Pancreas


- - 1 - - - - -

Adenoma: pars distalis 1 1 - 3 - 3 - - Pituitary Adenoma: pars intermedia - - - - - 1 - -

Skin Misc. Keratoacanthoma - - - - - 1 - - Hemangioma - - 1 - - - - - Hemangiosarcoma 1 - 3 - 1 1 - -

Spleen

Sarcoma (metastasis) - - 1 1 - - - - Adenoma 1 - - - - - - - Stomach Carcinoma (metastasis) - - - - - 1 - -





Papilloma - 1 - - - 1 - - Leiomyosarcoma (metastasis) - 1 - - - - - - Rhabdomyosarcoma (metastasis)

- - 1 - - - - -

Stomach Cont’d.

Sarcoma (metastasis) - - - 1 - - - - Hemangiosarcoma 1 - - - - - - - Fibrosarcoma 1 - - - - - 1 -

Subcutaneous Tissue


- - 1 - - 1

Tail Hemangioma 1 - - - - 1 - - Adenoma: interstitial cell 1 - 1 - 2 - - - Testis Adenoma: rete testis 2 - - - - - - - Adenoma: follicular cell 1 - - - - - - - Thyroid Carcinoma: follicular cell - 1 - - - 1 - -

Urinary Bladder Carcinoma: transitional cell 1 - - - - - - - Adenocarcinoma: endometrial - - - - - 1 - 1 Carcinosarcoma - - - - - - - 1

Uterus

Sarcoma: endomertrial stromal

- 1 - - - 2 - 1




Daily Dose: (mg/kg) 0 (Control) 100 300 600 Number evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions

Leiomyoma - - - 1 - - - 2 Leiomysarcoma - 2 - 2 - - - - Fibroma - - - 1 - - - - Carcinoma: squamous cell - - - - - 1 - - Benign granular cell Tumor - - - - - 1 - - Hemangioma - - - 1 - - - - Hemangiosarcoma - 1 - 2 - 2 - 1 Sarcoma (metastasis) - - - 1 - - - -

Uterus Cont’d.

Polyp: endometrial stromal - 6 - 5 - 3 - 4 Vagina Osteosarcoma (metastasis) - - - 1 - - - -




Daily Dose: (mg/kg) 0 (Control) 100 300 600 Number evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Noteworthy Findings Gross Pathology Duodenum Dilation 3 - 4 3 1 6 9 11 Thickening 4 1 2 2 3 2 5 9 Mass - - - - - - 1 2 Jejunum Dilatation 2 2 5 3 2 7 8 9 Ileum Dilatation 2 1 5 3 3 5 6 6 Cecum Dilatation 1 1 3 2 1 2 4 5 Liver Surface irregular - 2 1 2 - 5 4 9 Mass 13 2 4 3 - 2 5 10 Non-Neoplastic Lesions Bone Marrow Hypercellularity: myeloid 6 11 4 7 8 12 21 20 Duodenum Hyperplasia:

Villous/mucosal - 2 7 6 26 23 32 27

Kidney Karyomegaly: tubular - - - - - 1 3 8 Basophilic cell focus 3 1 - 1 - 3 - 4 Eosinophillic cell focus 2 - 1 2 3 7 8 8 Hypertrophy; Hepatocellular

9 6 14 6 22 27 39 46

Cyto/karyomegaly 16 2 9 4 21 27 35 36

Liver

Inclusions: nuclear 4 1 8 - 12 13 33 26 Necrosis: single cell 7 5 1 2 11 7 22 18 Proliferation: sinusoidal lining cells

3 2 3 3 7 15 20 25

Testis Mineralization: vascular - - 1 - 6 - 17 -




Daily Dose: (mg/kg) 0 (Control) 100 300 600 Number evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Tumor analysis Liver-hepatocellular adenomata - P=0.0037a Harderian gland P=0.0098a -

a Pair wise test

2.6.7.10.F. Carcinogenicity Report Title: An Oral Carcinogenicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Rat



2.6.7.10.F. R990204: An Oral Carcinogenicity Study of Tenofovir Disoproxil Fumarate (Tenofovir DF) in the Albino Rat

Species/ Strain: Rat, Sprague Dawley CD (Crl: CDR (SD) BR)- VAF

Duration of Dosing: 104 weeks Study No: R990204

Initial Age: 6 Weeks (approx) Method of Administration: Oral gavage Date of First Dose: 20 (Main) Vehicle/ Formulation: Suspension vehicle Treatment of Controls: Oral gavage (vehicle) GLP Compliance: Yes Basis for High-Dose Selection: Toxicity-based endpoint. The high dose was based on toxicity (GI tract toxicity, kidney tubular karyomegaly and bone toxicity) observed in a 13/42 week rat study. On day 10 the dose level for group 2 was increased to 300 mg/kg/day. Special Features: Separate populations for toxicokinetic evaluation were included in the study: Initial TK group (Set 1) comprised of 16 males and 16 females. Blood samples were taken on days 1 & 30. Animals euthanized on day 30 & subjected to necropsy. An additional group of TK animals were added to the study (Group 6, Set 2) to obtain plasma data from Day 1, 30, 60, and 90 at the revised dose level of 300 mg/kg/day. Animals euthanised on day 91 & subjected to necropsy. Additional populations of animals of 24 males and 24 females were added to Groups 1 to 4 to evaluate TK response for a period of 26 weeks (Set 3). Animals euthanized on day 182 & subjected to necropsy. Before assignment of animals to a treatment group, 10 males & 10 females animals, selected randomly, were subjected to a health screen. Daily Dose: (mg/kg) 0 (Control) 10/300# 30 100 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Toxicokinetics AUCss 0-24 ( g h/mL) (Day 180) - - 16.69 15.72 2.70 3.06 6.60 7.46 Number of Animals At Start 60 60 60 60 60 60 60 60 Died/Sacrificed Moribund 42 44 42 32 42 45@ 38 43 Terminal Sacrifice 18 16 18 28 18 15 22 17 Survival (%) 30 27 30 47 30 25 37 28 Body Weight (%) 644.8g 363.2g -5.5%* -3.7% +2.0% +0.6% -2.9% -1.1% Food Consumption (%) 206.4 144.0 -1.5% +5.6% -0.2% +2.8% -3.5% +3.9% # Dose increased on Day 10 from 10 to 300 mg/kg/day, * P 0.05 Dunnett’s, @ Terminated at Week 103




Daily Dose: (mg/kg) 0 (Control) 10/300# 30 100 Number evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions Abdomen Sarcoma - - - - - - 1 - Adrenal Adenoma: cortical - 2 - - 1 - - - Malignant

Pheochromocytoma 1 - - 2 1 - - -

Benign pheochromocytoma - - 3 1 1 1 3 - Metastasis - 1 - - - - - - Brain Malignant astrocytoma - 1 1 1 3 - 2 1 Malignant mixed glioma - - - - - - 1 - Metastasis 2 2 - 1 - 2 - 1 Benign granular cell Tumor - 1 - - - - - - Cavity

Cranial Metastasis - - - 1 - - - -

Clitoral Gland

Carcinoma: squamous cell - - - - - - - 1

Eye Malignant schwannoma - - - 1 - - - - Harderian

Gland Metastasis - - - 1 - - - -

Heart Metastasis - 1 - - - - - - # Dose increased on Day 10 from 10 to 300 mg/kg/day


Study No: CTBR 89282; R990204


Daily Dose: (mg/kg) 0 (Control) 10/300# 30 100 Number evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions Heart Benign schwannoma:

Endocardial - 1 - - - - 2 -

Hemolym. Malignant lymphoma - 2 1 - - 2 - 1 Tissue Histiocytic sarcoma 2 0 1 1 2 2 2 - Ileum Adenocarcinoma 1 - - - 1 - - - Jejunum Adenocarcinoma - - - - 1 - - - Kidney Carcinoma: tubular cell - - - - 1 - - - Adenoma: tubular cell 1 - - - - - - - Renal mesenchymal tumor - - - - 1 - - - Liver Carcinoma: hepatocellular 3 - 1 - 1 - 1 - Adenoma: hepatocellular 1 1 2 - - - - - Cholangioma - - - - - - 1 - Cholangiocarcinoma 1 - 1 - - - - - Metastasis 1 - - - - - - - Lung Carcinoma: metastasis - - 1 1 1 2 - 2 Adenoma:

Alveolar/bronchiolar - - 2 - 2 - 3 -

Metastasis - 1 - - - - - - Lymph node Metastasis 1 - - - 2 - - - # Dose increased on Day 10 from 10 to 300 mg/kg/day




Daily Dose: (mg/kg) 0 (Control) 10/300# 30 100 Number Evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions Lymph Node

Mandibular Metastasis - - - 1 - - - -

Mammary Fibroadenoma 1 23 2 25 1 23 - 20 Gland Adenoma - 3 - 2 - 1 - 2 Adenocarcinoma - 14 - 11 - 13 - 10 Nerve sciatic Metastasis - - - 1 - - - Ovary Benign luteoma - 1 - - - - - - Benign granulosa-theca

cell tumor - - - 2 - 2 - 2

Malignant schwannoma - - - 1 - - - - Pancreas Adenoma-acinar cell - - - - 1 - - - Carcinoma: islet cell 8 3 7 1 10 2 12 6 Adenoma: islet cell 4 - 6 2 5 2 2 1 # Dose increased on Day 10 from 10 to 300 mg/kg/day




Daily Dose: (mg/kg) 0 (Control) 10/300# 30 100 Number Evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions Parathyroid

Gland Adenoma - - - - 1 - - -

Pituitary Carcinoma: pars distalis 3 2 - 2 - 2 - 1 Adenoma: pars distalis 35 52 25 39 30 47 28 42 Adenoma: pars intermedia 3 - - 1 - - - - Skin Misc. Papilloma: squamous cell 1 - 1 - 3 - - - Carcinoma: squamous cell 1 2 1 - 1 - 1 1 Keratoacanthoma - - 1 1 1 - 4 - Adenoma: sebaceous - - - - - - 1 - Carcinoma: basal cell - 1 1 - - - 2 - Adenoma: basal cell - - - - - - 1 - Fibrosarcoma - - - - 1 - - - Histiocytoma - 1 - - - - - - Spleen Hemangioma - - - - 1 - - - Spinal Cord,

Cervical Benign astrocytoma - - - 1 - - - -

Subcutaneous Sarcoma - - 1 - - - - - Tissue Fibroma 2 1 2 - - - 2 - Fibrosarcoma 1 1 1 - - 1 - - Lipoma - - 3 2 - 2 - 1 Tail Fibrosarcoma 1 - - - - - 1 - Osteosarcoma - - - - - 1 - - # Dose increased on Day 10 from 10 to 300 mg/kg/day




Daily Dose: (mg/kg) 0 (Control) 10/300# 30 100 Number Evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Number of Animals with Neoplastic Lesions Testis Adenoma: intestitial cell 2 - 2 - 1 - - - Carcinoma: interstitial cell - - - - 1 - - - Adenoma: rete testis - - - - 1 - - - Thorax Liposarcoma - - - - 1 - - - Thyroid Adenoma: follicular cell 2 - 3 - - 1 1 1 Carcinoma: follicular cell - - - - 2 1 - - Adenoma: C-cell 4 4 4 6 2 3 3 2 Carcinoma: C-cell 2 1 1 - - - 1 - Tongue Papilloma: squamous cell - - - - 1 - 1 - Sarcoma - - - - - - - 1 Urinary

Bladder Papilloma: transitional cell - - - - - 1 1 -

Uterus Polyp: endometrial stromal - 2 - 11 - 4 - 3 Benign granular cell tumor - 3 - - - - - - Vagina Polyp - - - 1 - - - 1 Benign granular cell tumor - - - - - 1 - - Papilloma: squamous cell - - - 1 - - - - # Dose increased on Day 10 from 10 to 300 mg/kg/day




Daily Dose: (mg/kg) 0 (Control) 10/300# 30 100 Number Evaluated M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 M: 60 F: 60 Noteworthy Findings Gross Pathology - - - - - - - - Histopathology- Non-Neoplastic

Findings

Kidney Karyomegaly: tubular - - 29 17 1 - 5 - Chronic progressive

nephropathy 46 23 29 11 47 16 38 13

Tumor Analysis Uterus – Polyp:endometrial Stromal

Data P= 0.0053a P= 0.0065a

a Pair wise test # Dose increased on Day 10 from 10 to 300 mg/kg/day

2.6.7.11.A. Reproductive and Developmental Toxicity Nonpivotal Studies Test Article: Emtricitabine


2.6.7.11. Reproductive and Developmental Toxicity: Nonpivotal Studies 2.6.7.11.A. Emtricitabine

Species/ Strain


(Vehicle/ Formulation)

Dosing Period Doses mg/kg

No. per Group Noteworthy Findings

Study Number

Mouse / CD-1

Oral From gestation days 6–15

0, 250, 500, 1000, divided doses spaced by approximately six hours

5F/group One fetus in the control group had exencephaly without open eyelids (bilateral). No other external malformations and no developmental variations were observed in fetuses in this study. No other significant effect were reported. NOAEL = 1000 mg/kg/day for maternal and developmental toxicity of emtricitabine administered twice daily.

TOX033

Rabbit / New Zealand White

Oral From gestation days 7–19

0, 250, 500, 1000, divided doses spaced by approximately six hours

5F/group All animals survived to the scheduled necropsy on gestation day 29. No treatment-related clinical signs were observed at any dose level. Mean maternal body weights, body weight changes, net body weights, net body weight changes, gravid uterine weights, and food consumption were unaffected by treatment at all dose levels.Intrauterine growth and survival were also unaffected by treatment. Parameters evaluated included postimplantation loss, mean fetal body weights, viable litter size, and mean numbers of corpora lutea and implantation sites. No external malformations or developmental variations were observed in fetuses. No maternal toxicity or developmental toxicity was noted at dosage levels up to 1000 mg/kg/day.

TOX034

2.6.7.11.B. Reproductive and Developmental Toxicity Nonpivotal Studies Test Article: Rilpivirine



Species/Strain Sex/No. Per Group

Route/Method of

Administration (Vehicle/

Formulation) Duration of Dosing Doses (mg/kg/day) Batch no.

NOAEL (mg/kg/day) Noteworthy Findings

Study Number

Rat/Sprague Dawley 8 females/ group


GD6-17 0 (vehicle), 40, 120, 400 mg/kg/day TMC278 base

/ No effects TMC278-NC127

Rabbit/ New Zealand White 6 females/ group


GD6-19 0 (vehicle) , 25, 75, 150 mg/kg/day TMC278 base

/ 25 mg/kg/day: 1 F sacrificed during the study because of abortion of its litters. 75 mg/kg/day: 100% postimplantation loss. 150 mg/kg/day: 1F sacrificed during the study because of abortion of it’s litter, fecal output in all animals, body weight and food consumption, 100% post-implantation loss

TMC278-NC128

Rabbit/ New Zealand White 6 females/ group


GD6-19 0 (vehicle), 5, 20, 60 mg/kg/day TMC278 base

/ 5 mg/kg/day: No effects. 20 mg/kg/day: No effects. 60 mg/kg/day: Body weight, 2 F aborting prior to term, 2 F exhibiting total litter resorption (early resorptions) at term and 2 F with live litters exhibiting increased (33 and 43%) postimplantation loss.

TMC278-NC129

CA: citric acid; F: females; GD: gestation day; HPMC: hydroxypropylmethylcellulose; NOAEL: no observed effect level; PEG: polyethylene glycol; / = not determined

2.6.7.11.C. Reproductive and Developmental Toxicity Nonpivotal Studies Test Article: Tenofovir disoproxil fumarate



Species/ Strain



Dosing Period Doses mg/kg No. per Group Noteworthy Findings Study Number

Rat, Sprague Dawley/CD

Oral gavage

11 days, daily (days 7–17, gestation)

0, 30, 100, 300 or 1000 mg/kg/day

8F/group Mortality (1/8) in 1000 mg/kg/day on GD 12. maternal body weights and/or body weight gains, food consumption, ( 300 mg/kg/d). Urine stained fur, red perivaginal substance and emaciation in dams at 1000 mg/kg/day

fetal wgt at 1000 mg/kg/day No test article-related fetal gross observations or effects on other developmental parameters were observed.

97-TOX-4331-003 (dose ranging study)


Oral (Stomach Tube) 13 days, daily (days 6–18, gestation)

0, 30, 100 or 300 mg/kg/day

5F/group 300 mg/kg/day: liquid feces 1/5 does that aborted on GD 27. maternal body weight ( 100 mg/kg/d) and body weight gain (300 mg/kg/d),

absolute and relative feed consumption and maternal absolute kidney weights (300 mg/kg/d). No test article-related effect on Caesarean-sectioning or litter parameters; all fetuses appeared normal.

97-TOX-4331-006 (dose ranging study)

2.6.7.12.A. Reproductive & Developmental Toxicity: Fertility & Early Embryonic Development to Implantation

Report Title: Study of Fertility and Early Embryonic Development of TP-0006 Administered by Gavage to CD-1® Mice (Segment 1)



2.6.7.12. Reproductive & Developmental Toxicity: Fertility & Early Embroyonic Development to Implantation: 2.6.7.12.A. TOX036: Study of Fertility and Early Embryonic Development of TP-0006 Administered by Gavage to CD-1 ® Mice

(Segment 1)

Design similar to ICH 4.1.1 Duration of Dosing: M: received 4 weeks pre-breeding exposure, 21 days during mating and 21 days post-mating, for a total of 70 days F: 14 days prebreed, 21 days mating, 7 days post coitum (gestational day 0–6) for total maximum of 42 days.

Study Number: TOX036

Species/Strain: Mouse / CD-1® Day of Mating: 21 days Initial Age: 51 days old upon arrival Day of C-Section: on gestation day 13 Date of First Dose: M: or , 19 F: , 19

Method of Administration: Oral gavage, divided doses spaced by approximately six hours

GLP Compliance: Yes

Special Features: none Vehicle/Formulation: 0.5% methylcellulose 10 mL/kg/day

No Observed Adverse-Effect Level: F0 Males: The no observable adverse effect level (NOAEL) for systemic toxicity and for reproductive toxicity was at least 1000 mg/kg/day. F0 Females: The NOAEL for systemic toxicity and for reproductive toxicity was at least 1000 mg/kg/day. F1 Litters: The NOAEL for developmental toxicity was also at least 1000 mg/kg/day.

Conclusion: Exposure of F0 male and female mice by oral gavage during prebreeding and mating for both sexes and through implantation (on gestation day 6) for females, resulted in no toxicity for body weight or feed consumption. There were no effects of treatment on F0 male or female reproduction or on development of the F1 offspring through gestation day 13.





Daily Dose (mg/kg) 0 (Control) 250 500 1000 Males Toxicokinetics: AUC N/A NM NM NM No. Evaluated 21 20 21 21 Mortality No. Died 0/20 0/20 0/20 0/20 Sacrificed MoribundA 0 1 1 1 Clinical Observations N N N N Necropsy Observations Paired testes weight (g) 0.247 0.248 0.252 0.231 Paired epididymides weight (g) 0.116 0.119 0.120 0.111 Body Weight (%) N N N N Food Consumption (%) N N N N Reproductive Toxicity Mating index (%) 100 100 100 100 Fertility index (%) 96 90 100 90 Pregnancy index (%) 100 94.7 100 94.7 Epididymal sperm count (106/g) 620.1 692.2 667.3 757.2 Sperm motility (% motile) 69.3 68.9 69.7 71.1 A Three males were euthanized moribund due to injury or infection NM = Not measured N = Normal or no change from control group value.





Daily Dose (mg/kg) 0 (Control) 250 500 1000 Females Toxicokinetics: AUC N/A NM NM NM No. Evaluated 20 20 20 20 Mortality No. DiedA 0/20 0/20 0/20 0/20 Sacrificed moribund 0 0 0 0 Clinical Observations N N N N Necropsy Observations Gravid uterine weight (g) 6.65 6.92 6.30 6.59 Paired ovary weight (g) 0.049 0.055 0.064 0.058 Body Weight (%) N N N N Food Consumption (%) N N N N Reproductive Toxicity Mating index (%) 100 100 100 100 Fertility index (%) 96.0 90.0 100 90.0 Gestational index (%) 100 100 100 100 Preimplantation loss/litter (%) 5.74 4.08 11.46 9.04 Postimplantation loss/litter (%) 4.10 4.98 13.42 2.56 A No females died, were euthanized moribund, or were removed from study. NM = Not measured N = Normal or no change from control group value.





Dosage (mg/kg/day, two divided doses) 0 250 500 1000 Parents

Females that mated (Mating Index, %) 20 (100.0) 20 (100.0) 20 (100.0) 20 (100.0) Fertility Index (No. females pregnant/No. females mated, %) 96.0 90.0 100.0 90.0 Evaluated pregnant females 20 18 20 18

Litter Means (mean per litter ± standard deviation) Corpora lutea 13.25 0.42 13.22 0.46 14.10 0.69 14.06 0.89 Implantations (sites per litter) 12.50 0.34 12.89 0.42 12.35 0.50 12.61 0.52 Live fetuses (live implants per litter) 12.00 0.36 12.22 0.39 10.90 0.73 12.28 0.52 Dead fetuses (nonlive implants per litter) 0.50 0.14 0.67 0.18 1.45 0.45 0.33 0.11 Resorptions 0.45 0.14 0.61 0.16 1.35 0.45 0.33 0.11 Weight of fetuses (g) N/A N/A N/A N/A Sex ratio of fetuses N/A N/A N/A N/A

N/A = Not applicable

2.6.7.12.B. Reproductive & Developmental Toxicity: Fertility & Early Embryonic Development to Implantation

Report Title: A Fertility Study in Male Rats Given 524W91 by Gavage



2.6.7.12.B. TTEP/95/0028: A Fertility Study in Male Rats Given 524W91 by Gavage

Design similar to ICH 4.1.1 Duration of Dosing: M: 73 days (prior to mating and throughout mating – dose days 1–95) F: untreated

Study Number: TTEP/95/0028

Species/Strain: Rat / CD (Sprague-Dawley)

Day of Mating: Bred-dams day 0 (date of insemination)

Initial Age: M: F0 males approx. 17 weeks of age at time of pairing, F: F0 approx. 12 weeks of age at time of pairing

Day of C-Section: Calculated gestation day 13 cesarean section

Date of First Dose: M: , 19 F: , 19

Method of Administration: Oral gavage GLP Compliance: Yes

Special Features: none Vehicle/Formulation: 524W91 + 0.5% methylcellulose, 20 mL/kg/dose

No Observed Adverse-Effect Level: F0 Males: NOEL = 750 mg/kg/day, NOAEL = 3000 mg/kg/day F0 Females: 3000 mg/kg/day F1 Litters: 3000 mg/kg/day

Conclusion: Male rats given 150, 750 or 3000 mg 524W91/kg/day orally for 73 days prior and throughout the period of mating to untreated female rats exhibited postdose salivation primarily at the high dose, but did not show any other evidence of treatment-related toxicity. No adverse effects on fertility, reproductive performance, sperm count, and sperm motility or pathological changes in the reproductive organs were observed.





Results for F0 Generation Males: Daily Dose (mg/kg) 0 (Control) 150 750 3000 No. Evaluated 25 25 25 25 Mortality 0 0 0 1 (dosing accident) Clinical Observations None None Transient postdose

salivation Postdose salivation

Body weight (%) - No change No change No change Food consumption (%) - Normal Normal Normal Necropsy Observations Histopathology 1/10 nephritis

2/10 prostatitis - - 3/10 prostatitis

Gross lesions 1a 0 0 0 Organ weight (%) - No change No change No change Sperm count and motility - Normal Normal Normal NM = Not measured N = Normal or no change from control group value. a Raised white areas on the kidneys





Results for F0 Generation Untreated Females: Daily Dose (mg/kg) 0 (Control) 150 750 3000 No. Evaluated 25 25 25 25 Body weight change (gestation) - No change No change No change Food consumption (gestation) - No change No change No change Maternal gross lesionsA None None None None Reproductive performance: Fertility index, % 87.5 96.0 92.0 100.0 Pregnancy rate, % 88.0 96.0 92.0 100.0 Number of pregnant females 22 24 23 25 Total resorption 0 1 0 0 Implantation sites/dam 15.6 15.6 17.3 17.0 Viable fetuses/dam 14.4 14.8 15.7 16.2 Post-implantation loss/dam 1.2 0.8 1.6 0.8 NM = Not measured N = Normal or no change from control group value. A Raised white areas on kidney





Dosage (mg/kg/day, two divided doses) 0 150 750 3000 Parents

Females with sperm (Fertility index, %) 24 (87.5) 25 (96.0) 25 (92.0) 25 (100.0) Pregnant females (Pregnancy rate, %) 21 (88.0) 24 (96.0) 23 (92.0) 25 (100.0) Evaluated pregnant females 21 24 23 25 Litter Means (mean per litter ± standard deviation) Corpora lutea 18.2 3.4 17.4 3.9 18.3 1.8 19.0 2.4 Implantations 15.6 3.5 15.6 5.2 17.3 1.7 17.0 1.5 Live fetuses 14.4 3.4 14.8 5.3 15.7 2.9 16.2 1.6 Dead fetuses N/A N/A N/A N/A Resorptions 1.2 1.3 0.8 1.2 1.6 2.0 0.8 0.9 Weight of fetuses (g) N/A N/A N/A N/A Sex ratio of fetuses N/A N/A N/A N/A

N/A = Not applicable

2.6.7.12.C Reproductive & Developmental Toxicity: Fertility & Early Embryonic Development to Implantation

Report Title: Oral (Gavage) Male Fertility Study in the Rat



2.6.7.12.C. TMC278-NC124: Oral (Gavage) Male Fertility Study in the Rat

Species/strain: Rat/Sprague-Dawley Age/weight at first dose: 6-7 weeks (M, first dose), 9-10 weeks (F, mating) Duration of dosing: 14 weeks Duration of postdose: 0 days

Administration route/method: Oral/Gavage Doses: 0 (vehicle), 100, 400, 1600 mg base eq./kg/day TMC278.HCl Test article batch:

Vehicle/formulation: 0.5% w/v HPMC

Testing facility: Study No.: TMC278-NC124 GLP Compliance: Yes Date start of study: 20

No Observed Adverse Effect Level: 1600 mg base eq./kg/day Toxicokinetics: Not determined Noteworthy Findings: Dose (mg base eq./kg/day) 0 100 400 1600 No. of animals 25M 25F 25M 25F 25M 24F 25M 25F Died or killed prematurely - - - - - - - - Clinical observations - / - / - / - / Body weight (g) - / - / - / - / Food consumption (g) - / - / - / - / Copulation index (%) - - - - - - - - Fertility index (%) - - - - - - - - Sperm motility (%) - / - / - / - / Sperm morphology - / - / - / - / Mean no. corpora lutea / - / - / - / - Pre-implantation loss (% of corpora lutea) / - / - / - / - Post-implantation loss (% of implantation sites) / - / - / - / - Mean gravid uterus weight / - / - / - / - Organ weights - / - / - / - /

F: female; HPMC: hydroxypropylmethylcellulose; M: male; - no noteworthy findings. /: not done.

2.6.7.12.D Reproductive & Developmental Toxicity: Fertility & Early Embryonic Development to Implantation

Report Title: Oral (Gavage) Female Fertility Study in the Rat



2.6.7.12.D. TMC278-NC125: Oral (Gavage) Female Fertility Study in the Rat

Species/strain: Rat/Sprague-Dawley Age at first dose: 11 - 12 weeks Duration of dosing: 14 days prior to pairing + 7 days of pregnancy Duration of post-dose: None

Administration route/method: Oral/gavage Doses:0, 40, 120, 400 mg base eq./kg/day TMC278.HCl Test article batch: Vehicle/formulation: 0.5% w/v HPMC

Testing facility: J&J PRD Study No.: TMC278-NC125 GLP compliance: Yes Date start of study: 20

No Observed Adverse Effect Level: 400 mg base eq./kg/day Dose (mg base eq./kg/day) 0 (vehicle) 40 120 400

No. of animals F: 24 F: 24 F: 24 F: 24

Mortality/Sacrifice 0/24 0/24 0/24 0/24 Behavior and appearance N N N N Body weight gain N N N N Corrected maternal weight gain N N N N Food consumption N N N N Weight of gravid uterus N N N N Gross pathology N N N N Pre-coital interval N N N N Copulation rate N N N N Fertility rate N N N N Implantations N N N N Corpora lutea of pregnancy N N N N Pre-implantation loss N N N N Embryos N N N N Resorptions N N N N Post-implantation loss N N N N F: female; HPMC: hydroxymethylcellulose; N: no relevant changes

2.6.7.12.E. Reproductive & Developmental Toxicity: Fertility & Early Embryonic Development to Implantation

Report Title: Oral (Gavage) Fertility and General Reproduction Toxicity Study of GS-4331-05 (bis-POC PMPA) in Sprague-Dawley Rats


A = One animal found dead on day 54. Death attributed to intubation accident; B = Terminal weights for control groups (means) are shown. For treatment groups, % differences from controls are shown; C = Excludes values for rats not assigned to cohabitation, because there were no female rats; D = Excludes 1 male rat cohabitated with a female rat that was sacrificed on day 17. E= Includes only one confirmed mating for each male rat; F= Includes only 1 pregnancy for each rat that impregnated 1 female * =p 0.05, ** =p 0.01 CONFIDENTIAL Page 271 17AUG2010

2.6.7.12.E. 98-TOX-4331-006: Oral (Gavage) Fertility and General Reproduction Toxicity Study of GS-4331-05 (bis-POC PMPA) in Sprague-Dawley Rats

Design Similar to ICH 4.1.1 Duration of Dosing:

M: 28 days prior to cohabitation, through cohabitation (maximum 21 days) & continuing through to the day before sacrifice.

Study No: 98-TOX-4331-006

Species. Strain: Rat Crl: CD BR VAF/Plus (Sprague Dawley)

F: 15 days prior to cohabitation, through cohabitation (maximum 21 days) through to day 7 of presumed gestation.

Initial age: M: 57 days, F: 65 days Day of mating: Day 28 GLP Compliance: Yes Date of First Dose: M: 19 , F:

19 Day of C-section: Day 20 presumed gestation.

Special Features: None Method of Administration: Oral gavage NOEL: F0 Males: 100mg/kg. Vehicle Formulation: Suspension vehicle F0 Females: 100 mg/kg Reproductive: 300 mg/kg Developmental: 600 mg/kg Toxicokinetics: Not determined Daily Dose (mg/kg) 0 Control 100 300 600 Males No. Evaluated 25 25 25 25 Mortality 0 0 1A 0 No died 0 0 1A 0 Moribund sacrificed 0 0 0 0 Clinical Observation Maximal possible incidence

(days x rats)/No of rats per group 1612/25 1612/25 1602/25 1613/25

Excess salivation 0/0 10/8* 14/8* 53/17** Dry, red perioral substance 0/0 1/1 3/3 23/7**




A = One animal found dead on day 54. Death attributed to intubation accident; B = Terminal weights for control groups (means) are shown. For treatment groups, % differences from controls are shown; C = Excludes values for rats not assigned to cohabitation, because there were no female rats; D = Excludes 1 male rat cohabitated with a female rat that was sacrificed on day 17. E= Includes only one confirmed mating for each male rat; F= Includes only 1 pregnancy for each rat that impregnated 1 female * =p 0.05, ** =p 0.01 CONFIDENTIAL Page 272 17AUG2010

Daily Dose (mg/kg) 0 Control 100 300 600

Males Necropsy Observations (cont) Body Weight (%)B 558.6g 0 0 0 Kidney Weight (paired)B 4.4154g 0 -1** -1* Mating & Fertility Rats in Cohabitation 25 25 25 25 No in Analysis 25 25 23C, D 23C Days in Cohabitation mean

(SD) 2.6 (1.1) 3.5 (3.4) 3.0 (2.4) 2.4 (1.0)

No of Males that Mated (%) 25 (100) 23 (92) 23 (100) 23 (100) Fertility Index

Pregnancies/No. Mated (%) 23/25 (92) 23/23 (100) 22/23 (95.6) 22/23 (95.6)

Male Rats with Confirmed Mating DatesE

25 22 23 23

Male Rats Mating Times Days 1–7 25 22 22 23 Days 8–14 0 0 1 0 Rat Pregnant/Rats in

Cohabitation F (%) 23/25 (92) 23/25 (92) 22/23 (95.6) 22/23 (95.6)




A = One animal found dead on day 54. Death attributed to intubation accident; B = Terminal weights for control groups (means) are shown. For treatment groups, % differences from controls are shown; C = Excludes values for rats not assigned to cohabitation, because there were no female rats; D = Excludes 1 male rat cohabitated with a female rat that was sacrificed on day 17. E= Includes only one confirmed mating for each male rat; F= Includes only 1 pregnancy for each rat that impregnated 1 female * = p 0.05, ** = p 0.01 CONFIDENTIAL Page 273 17AUG2010

Daily Dose (mg/kg) 0 Control 100 300 600 Females No. Evaluated 25 25 25 25 Mortality 0 0 2 2 No died 0 0 0 1A Moribund Sacrificed 0 0 2 1 Clinical Observation Maximal Possible Incidence

(days x rats)/No of Rats per Group

375/25 375/25 375/25 367/25

Dry (Brown or Red) Perioral Substance

0/0 0/0 3/3C 32/14**E, F

Excess Salivation 0/0 0/0 2/2C, D 12/7**E Urine-Stained Abdominal Fur 0/0 0/0 0/0 8/3**E Necropsy Observations Body Weight (%)B 411.3g 0 0 0 Mating & Fertility Predose Estrous Estrous Stages 14 Days

mean (SD) 3.2 (0.6) 2.9 (0.9) 3.2 (0.7) 2.9 (0.8)

Precohabitation Estrous Cycling Estrous Stages 14 Days

mean (SD) 3.4 (0.7) 3.3 (0.5) 3.1 (0.7) 22.3 (0.9) **




A = One animal found dead on day 54. Death attributed to intubation accident; B = Terminal weights for control groups (means) are shown. For treatment groups, % differences from controls are shown; C = Excludes values for rats not assigned to cohabitation, because there were no female rats; D = Excludes 1 male rat cohabitated with a female rat that was sacrificed on day 17. E= Includes only one confirmed mating for each male rat; F= Includes only 1 pregnancy for each rat that impregnated 1 female * = p 0.05, ** = p 0.01 CONFIDENTIAL Page 274 17AUG2010


Females Rats in Cohabitation 25 25 23 23 (cont) Days in Cohabitation

(mean SD) 2.6 (1.1) 3.7(3.8) 3.0(2.4) 2.4(1.0)

Rats that Mated (%) 25 (100) 25(100) 23(100) 23(100) Fertility Index

Pregnancies/No. Mated (%) 23/25 (92) 25/25 (100) 22/23 (95.6) 22/23 (95.6)

Rats with Confirmed Mating Dates

25 24 23 23

Rats Pregnant/Rats in Cohabitation.

23/25 (92) 25/25 (100) 22/23 (95.6) 22/23(95.6)

Litter Mean per Litter Corpora lutea 18.4 17.8 18.2 17.3 Implantations 15.3 15.5 16.0 15.1 Live Fetuses 14.7 15.1 15.2 14.7 Dead Fetuses 0 0 0 0 Resorptions 0.6 0.4 0.8 0.4 M F M F M F M F Weight of Fetuses (g) 3.69 3.47 3.75 3.56 3.78 3.57 3.79 3.63 Sex Ratio of Fetuses 50 50 48.4 51.6 49.1 50.9 49.8 50.2

2.6.7.13.A. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: A Study of the Effects of TP-0006 (FTC) on Embryo/Fetal Development in Mice



2.6.7.13. Reproductive & Developmental Toxicity: Effects on Embryo-fetal Development 2.6.7.13.A. TOX037: A Study of the Effects of TP-0006 (FTC) on Embryo/Fetal Development in Mice

Design similar to ICH 4.1.3 Duration of Dosing: F: From gestation Days 6–15

Study No. TOX037

Day of Mating: Day 0 Species/Strain: Mouse / CD-1 Day of C-Section: Gestation day 18 Initial Age: 72 day old upon receipt Method of Administration: Oral,

divided doses spaced by approximately six hours

Date of First Dose: 19 Vehicle/Formulation: TP-0006 (FTC) +0.5% methylcellulose 10 mL/kg/day

GLP Compliance: Yes

Special Features: none No Observed Adverse-Effect Level: NOAEL = 1000 mg/kg/day for maternal and developmental toxicity of emtricitabine administered twice daily

F0 Females: F1 Litters:

No material toxicity or developmental toxicity was noted in this study.

2.6.7.13.A. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: A Study of the Effects of TP-0006 (FTC) on Embryo/Fetal Development in Mice



Daily Dose (mg/kg) 0 (Control) 250 500 1000

F0 ToxicokineticsA: Conc. mean ± stdev. g/mL N/A 52 15 88 14 186 14

Females Females with Sperm ND ND ND ND Pregnant Females 23 25 23 22 Evaluated Pregnant Females 23 25 23 22

Litters No. Corpora Lutea 15.3 3.59 15.3 2.69 14.3 2.37 14.9 3.00 No. Implantations 12.3 2.42 12.6 2.58 11.9 3.23 12.1 2.47 Live Fetuses 11.4 2.68 11.7 2.84 11.0 3.20 11.7 2.36 Dead Fetuses 0 0 0.0 0.21 0.0 0.21 Resorptions (early) 0.8 1.17 0.8 1.09 0.7 0.70 0.4 0.58 Resorptions (late) 0.1 0.29 0.2 0.37 0.1 0.34 0.0 0.21 Weight of Fetuses (g) 1.34 0.088 1.34 0.113 1.34 0.117 1.26 0.102 Sex of Fetuses (% of live fetuses, m|f) 48.0 52.0 51.8 48.2 47.6 52.4 49.8 50.2 Litters with any Fetal Alteration (%) 1 (1) 4 (3) 1 (1) 3 (3) Fetuses available for Morphological Evaluation (%) 263 (23) 292 (25) 254 (23) 257 (22)

N/A = Not Applicable, ND = Not determined, Conc = Concentration, stdev = standard deviation A A toxicokinetic phase was conducted with 5 female mice/group administered 250, 500, and 1000 mg/kg/day as a single dose on gestation day 15. The mean standard

deviation for plasma concentrations were determined. The increase in plasma levels was roughly in proportion to the increase in dose administered at 0.5 hr.

2.6.7.13.B. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: A Study of the Effects of TP-0006 on Embryo/Fetal Development in Rabbits



2.6.7.13.B. TOX038: A Study of the Effects of TP-0006 on Embryo/Fetal Development in Rabbits

Design similar to ICH 4.1.3 Duration of Dosing:

F: From gestation days 7–19 Study No. TOX038

Day of Mating: Day 0 Species/Strain: Rabbit/New Zealand White Day of C-Section: gestation day 29 Initial Age: approximately 4.5 months old Method of Administration: Oral,

divided doses spaced by approximately six hours

Date of First Dose: , 19 Vehicle/Formulation: TP-0006 (FTC) + 0.5% methylcellulose, 10 mL/kg/day

GLP Compliance: Yes

No Observed Adverse-Effect Level: F0 Females: A NOAEL of 100 mg/kg/day was established for maternal toxicity. F1 Litters: A NOAEL of 1000 mg/kg/day was considered for developmental toxicity of TP-0006 when administered twice

daily to rabbits

Emtricitabine was rapidly absorbed with Cmax occurring generally within 1 hour post-dose. AUC and Cmax increased linearly with dose. Plasma elimination t1/2 was 3 4 hours at all dose levels. Fetal/maternal exposure ratios were around 0.4–0.5 one hour after dosing (at tmax) for all dose levels. Emtricitabine is readily transferred across the placenta.

2.6.7.13.B. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: A Study of the Effects of TP-0006 on Embryo/Fetal Development in Rabbits




F0 ToxicokineticsA: AUC0-12 ( g.h/mL) N/A 43.6 157.6 628.9 AUC0-24 ( g.h/mL) N/A 87.3 315.2 1257.8

Females Females with Sperm ND ND ND ND Pregnant Females 18 19 19 17 Evaluated Pregnant Females 17 18 18 16

Litters (means per litter standard deviation) No. Corpora Lutea 9.9 1.73 10.4 2.31 10.2 2.29 12.1 3.86 No. Implantations 8.1 2.37 7.6 2.52 7.2 2.04 7.0 2.90 Live Fetuses 7.6 2.37 6.8 3.01 6.2 2.60 6.9 2.70 Dead Fetuses 0 0 0 0 Resorptions (early) 0.4 0.70 0.7 1.28 0.8 1.76 0.1 0.25 Resorptions (late) 0.2 0.39 0.1 0.32 0.1 0.32 0.1 0.25 Weight of Fetuses (g) 44.4 4.86 45.7 6.44 47.0 5.72 42.4 5.77 Sex of Fetuses (% of live fetuses, m|f) ND ND ND ND Litters with Any Fetal Alteration (%) 0 0 0 0 Fetuses Available for Morphological Evaluation (%) 129 (17) 123 (17) 112 (18) 110 (16)

ND = Not determined N/A = Not Applicable A Additional evaluations: On gestation day 19, satellite groups of 5 rabbits per dose were used to provide plasma samples to evaluate drug absorption. Animals were bled pre-

dose, 1, 3, 7, and 12 hours after the first dose (the second dose was withheld until after plasma collection).

2.6.7.13.C. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: Oral Developmental Toxicity Study in the Rat



2.6.7.13.C. TMC278-NC105: Oral Developmental Toxicity Study in the Rats

Species/strain: Rat/SPF Sprague Dawley Age/weight: about 11-12 weeks/245 - 301 g Duration of dosing: Gestation Days (GD) 6-17 Day of mating: GD0 Day of c-section: GD21

Administration route/method: Oral/Gavage Doses: 0, 40, 120 and 400 mg/kg/day TMC278 base Test article batch: Vehicle/formulation: PEG400 + CA

Testing facility: J&J PRD Study No.: TMC278-NC105 GLP Compliance: Yes Date start of study: 20

No Observed Adverse Effect Level: 40 mg/kg/day (maternal and embryo-fetal) Toxicokinetics:

Doses in mg/kg/day 0 (vehicle) 40 120 400

No. of animals - F:18 F:18 F:18 Cmax ( g/mL) GD6 - 4.9 6.0 14 Cmax ( g/mL) GD16 - 5.6 7.2 13 AUC0- ( g.h/mL) GD6 - 33 65 182 AUC0-24h ( g.h/mL) GD16 - 37 63 152

Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 40 120 400

No. of animals 24F 24F 24F 24F Mortality 0 0 0 0 Pregnancy rate 21/24 24/24 23/24 23/24 Behavior and appearance N N N N Body weight gain N N (+)D (+)D (GD10-13) Corrected maternal weight gain N N (+)D (+)D Food consumption N N +D (GD10-17) +D (GD10-17) Weight of gravid uterus N N N N Thyroid weight N N +I +I Gross pathology N N N N

CA: citric acid; N = no relevant changes; D = decreased; F: female; I = increase; (+) = minimal; + = slight; GD = gestational day; PEG400: plyethylene glycol 400

2.6.7.13.C. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: Oral Developmental Toxicity Study in the Rat



Study TMC278-NC105: Oral Developmental Toxicity Study in the Rats (continued) Noteworthy Findings: Dose (mg/kg/day) 0 (vehicle) 40 120 400

No. of animals 24F 24F 24F 24F

Litter Data Implantations N N N N Corpora lutea of pregnancy N N N N Pre-implantation loss N N N N Live fetuses N N N N Dead fetuses N N N N Mean litter size N N N N Early resorptions N N N N Late resorptions N N N N Post-implantation loss N N N N Body weight of live fetuses N N N N Sex ratio N N N N Fetal observations Dilated renal pelvis N N +I +I N = no relevant changes; I = increase; + = slight; F: female.

2.6.7.13.D. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: Oral Developmental Toxicity Study in the Rabbit



2.6.7.13.D. TMC278-NC130: Oral Developmental Toxicity Study in the Rabbit

Species/strain: Rabbit/New Zealand white Age at first hormone injection/weight at first dose: between 18-21 weeks/2869 to 3794 g Duration of Dosing: GD6 - 19 Day of Mating: GD0 Day of C-Section: GD28

Administration route/method: Oral/gavage Doses: 0, 5, 10 and 20 mg/kg/day, as TMC278 base Test article batch: Vehicle/formulation: 0.5% w/v HPMC

Testing facility: J&J PRD Study No.: TMC278-NC130 GLP Compliance: Yes Date start of study: 20

Special features: None No Observed Adverse Effect Level: 20 mg/kg/day (maternal) and 10 mg/kg/day (fetal). Toxicokinetics:

Doses in mg/kg/day 0 (vehicle) 5 10 20 No. of animals - F:3 F:3 F:3 Cmax ( g/ml) GD6 - 6.4 9.7 13 Cmax ( g/ml) GD19 - 6.7 10 15 AUC0- ( g.h/ml) GD6 - 95 162 219 AUC0-24h ( g.h/ml) GD19 - 105 170 232

Dose (mg/kg/day) 0 5 10 20

No. of animals F:20 F:20 F:20 F:20 Mortality - 0/20 0/20 0/20 Clinical observations - N N N Pregnancy ratea - 16/20 14/20 18/20 Body weight gain - N N N Corrected mean maternal weight gain - N N N Food consumption - N N N Weight of gravid uterus - N N N Adrenal weights - N N N Gross pathology - N N N

a Number of pregnant females/ terminally sacrificed females; HPMC: hydroxypropylmethylcellulose; N = no relevant changes, GD = gestational day

2.6.7.13.D. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: Oral Developmental Toxicity Study in the Rabbit



Study: TMC278-NC130: Oral Developmental Toxicity Study in the Rabbit (continued) Dose (mg/kg/day) 0 5 10 20

No. of animals F:20 F:20 F:20 F:20

Litter data Implantations - N N N Corpora lutea of pregnancy - N N N Pre-implantation loss - N N N Live fetuses - N N N Dead fetuses - N N N Mean litter size - N N N Early resorptions - N N N Late resorptions - N N N Post-implantation loss - N N N Weight of live fetuses - N N N Sex ratio - N N N Fetal observations left subclavian artery branches arising from the aorta

- N N +I

hypoplastic interparietal bone - N N +I I = increase; + = slight; N = no relevant changes.

2.6.7.13.E. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: Oral (Gavage) Developmental Toxicity Study of GS-4331-005 in Rats


*=p 0.05, **=p 0.01. a = Terminal weights for control groups are shown. For treatment groups, % differences from controls are shown CONFIDENTIAL Page 283 17AUG2010

2.6.7.13.E. 97-TOX-4331-004: Oral (Gavage) Developmental Toxicity Study of GS-4331-005 in Rats


Days 7- 17 of presumed gestation Study No: 97-TOX-4331-004

Species. Strain: Rat Crl: CD BR VAF/Plus (Sprague Dawley)

Initial age: 65 Days Day of mating: Day 0 GLP Compliance: Yes Date of First Dose: 19 Day of C-section: Day 20 presumed gestation Special Features: None Method of Administration: Oral gavage NOEL: Fo Females. 150 mg/kg Vehicle Formulation: Suspension vehicle F1 Litters 450 mg/kg


Fo Toxicokinetics: No toxicokinetic data were measured Females No. Evaluated 25 25 25 25 Mortality Number died 0 0 0 0 Moribund sacrificed 0 0 0 0 Clinical Observation Maximal possible

incidence (days x rats)/No of rats per group

350/25 350/25 350/25 350/25

Localized Alopecia Underside

0/0 0/0 0/0 34/4**

Necropsy Observations Body weight (%) a 414.2g 0 0 0

2.6.7.13.E. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: Oral (Gavage) Developmental Toxicity Study of GS-4331-005 in Rats




Females Females with sperm 25 25 25 25 (Cont) Pregnant females 24 23 25 24 Evaluated pregnant females 24 23 25 23 Litters Mean per litter Corpora lutea 15.9 15.7 15.5 14.9 Implantations 15.0 14.6 14.6 13.9 Live fetuses 14.4 14.1 14.1 13.4 Dead fetuses 0 0 0 0 Resorptions 0.5 0.5 0.6 0.4 M F M F M F M F Weight of fetuses (g) 3.81 3.62 3.86 3.63 3.88 3.59 3.84 3.61 Sex ratio of fetuses 51.8 48.2 49.8 50.2 51.8 48.2 49.5 50.5 Litters with any fetal alteration

(%) 45.8 39.1 28.0 21.7

Fetuses with any alteration (%) 5.5 4.9 2.8 1.6

2.6.7.13.F. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: Oral (Stomach Tube) Developmental Toxicity Study of GS-4331-05 in Rabbits



2.6.7.13.F. 98-TOX-4331-005: Oral (Stomach Tube) Developmental Toxicity Study of GS-4331-05 in Rabbits


Days 6-18 of presumed gestation Study No: 98-TOX-4331-005

Species. Strain: Rabbit, New Zealand White [Hra: NZW) SPF]

Initial age: 5 months Day of Mating: Day 0 GLP Compliance: Yes Date of First Dose: 19 Day of C-section: Day 29 Special Features: None Method of Administration: Oral gavage NOEL: F0 Females. 100 mg/kg Vehicle Formulation: - F1 Litters 300 mg/kg Daily Dose (mg/kg) 0 Control 30 100 300

Females Toxicokinetics (Determined in study 98-TOX-4331-005PK) AUC (0- ) g*hr/mL

Day 18 presumed gestation (n = 4, mean (SD)

- 19.15 (4.4) 65.30 (16.07) 212.48 (97.14)

No. Evaluated 35 35 25 35 Mortality Number died 0 0 0 0 Moribund sacrificed 0 0 0 1B Clinical Observation Maximal possible incidence

(days x rabbits)/No of rabbits per group

800/35 800/35 560/25 800/35

Scant feces 0/0 0/0 0/0 20/9** Localised Alopecia (Limbs) 4/1 0/0 6/3** 0/0 Necropsy observations Body weight (%) a, 3963g 0 0 0

2.6.7.13.F. Reproductive & Developmental Toxicity: Effects on Embryofetal Development

Report Title: Oral (Stomach Tube) Developmental Toxicity Study of GS-4331-05 in Rabbits




Females Females with sperm 35 35 25 35 (Cont) Pregnant females 27 28 23 25 Evaluated pregnant females 23 24 20 20 Litters Mean per litter Corpora lutea 10.0 10.2 9.3 10.9 Implantations 7.4 7.2 6.0 8.2 Live fetuses 7.0 6.9 5.8 7.6 Dead fetuses 0 0 0 0 Resorptions 0.4 0.3 0.2 0.6 M F M F M F M F Weight of Fetuses (g) 47.81 46.42 47.96 46.19 49.74 46.75 46.19 44.22 Sex Ratio of Fetuses 55.1 44.9 44.8 55.2 57.9 42.1 39.0 61.0

2.6.7.14.A. Reproductive & Developmental Toxicity: Effects on Pre- & Post-Natal Development: Including Maternal Function

Report Title: Pre- and Postnatal Development Study in CD-1® Mice Given TP-0006 by Gavage (Segment III)



2.6.7.14. Reproductive & Developmental Toxicity: Effects on Pre- & Post-Natal Development, Including Maternal Function

2.6.7.14.A. TOX039: Pre- and Postnatal Development Study in CD-1® Mice Given TP-0006 by Gavage (Segment III)

Design similar to ICH 4.1.2 Duration of Dosing:

F: Gestation day 6 to postnatal day 20 Study No. TOX039

Day of Mating: Fo Gestation day (Fo GD 0) and F1 GD 0

Species/Strain: Mouse / CD-1 Method of Administration: Oral, divided doses spaced by approximately six hours

Initial Age: 58 days old upon arrival Vehicle/Formulation: TP-0006 (FTC) +0.5% methylcellulose 10 mL/kg/day

Date of First Dose: , 19 Litters Culled/Not Culled GLP Compliance: Yes Special Features: none No Observed Adverse-Effect Level:

F0 Females: NOEL was 1000 mg/kg/day for F0 maternal toxicity and F0 reproductive toxicity F2 F1 Males: NOEL was 1000 mg/kg/day for postwean F1 male toxicity F1 Females: NOEL was 1000 mg/kg/day for postwean F1 female toxicity, F1 reproductive toxicity F2 and developmental

toxicity





Daily Dose (mg/kg) 0 (Control) 250 500 1000 F0 Toxicokinetics: AUC N/A ND ND ND Females

Females with Sperm 25/25 25/25 24/25A 25/25 Pregnant Females 24 24 24 25 Females with Delivery 24 24 22B 25

F0 Maternal Toxicity Mortality (number): Gestation 0 0 0 0

Lactation+ 0 2 0 2 Gestational Body Weights No toxicity Gestational Weights Changes No toxicity Gestational Feed Consumption No toxicity Gestational Clinical Observations No toxicity Lactational Body Weights No toxicity Lactation Weight Changes No toxicity Lactational Feed Consumption No toxicity Lactational Clinical Observations No toxicity F0 Reproductive Indices No toxicity

Parental Gross Necropsy F0 Female Terminal Body Weights and Findings No toxicity

ND = Not determined N/A = Not Applicable A Female 3 was removed due to a dosing error (misdirected dose) on gestational day 12. B Female 6 had implant sites only and was included as a pregnant female that did not have a live litter. Female 14 had started to deliver but had no pups the next morning. She

was noted as a pregnant female but was not included in the live litter since it was not observed if live pups were present when she was in the process of delivering. + The causes of 4 deaths were not determined; the lack of a dose-response relationship indicates that they were not treatment-related.





Daily Dose (mg/kg) 0 (Control) 250 500 1000 Litters

Implantation Sites per Litter 12.54 0.41 12.83 0.52 12.25 0.37 12.12 0.54 Live Births 12.54 0.48 12.63 0.47 12.91 0.41 12.76 0.29 Stillbirths 0.04 0.04 0 0.05 0.05 0 Survivors Index pnd 4C 94.93 4.16 94.66 4.15 98.67 0.62 97.92 0.79 Lactation Index pnd 21D 97.75 1.05 98.21 1.30 99.43 0.57 98.91 0.75 Weight at Birth (g) – Male 1.60 0.03 1.62 0.03 1.56 0.02 1.63 0.02 Weight at Birth (g) – Female 1.56 0.03 1.55 0.03 1.52 0.02 1.55 0.02 Weight at Weaning (g) – Male 13.97 0.32 14.21 0.29 13.22 0.21 14.01 0.29 Weight at Weaning (g) – Female 13.51 0.29 13.50 0.27 12.70 0.22 13.33 0.28 Sex ratio of Live Newborns (% of live newborns m|f) 47.0 53.0 48.1 51.9 49.6 50.4 49.2 50.8

ND = Not determined N/A = Not Applicable pnd = postnatal day C Number surviving 4 days/number live on pnd 0 D Number surviving 21 days/number live on pnd 4





Daily Dose (mg/kg) 0 (Control) 250 500 1000 F1 Offspring Toxicity

Lactational Survival (number of litters died) 1 1 0 0 Postwean Developmental Landmarks: Estrous Cyclicity N N N ** Postwean Body Weights N N **a N Postwean Body Weight Changes N N **b *c Lactational Body Weights/Litter No toxicity Lactational Sex Ratio/Litter No toxicity Lactational Clinical Observations No toxicity Postwean Developmental Landmarks (vaginal patency, pupillary reflex, motor activity, learning and memory, postwean clinical observations)

No toxicity

F1 Reproductive Toxicity Reproductive Indices No toxicity Gestational Body Weights No toxicity Gestational Body Weight Changes No toxicity Gestational clinical observations No toxicity Lactational body weights (pnd 0 and 1) No toxicity Lactation body weight changes No toxicity Lactational clinical observations No toxicity

Parental Gross Necropsy F0 male terminal body weights N N * N F0 female terminal body weights and findings No toxicity F0 male findings No toxicity

N Normal; no statistically significant or biologically relevant change. or Increase or decrease * p 0.05 versus control ** p 0.01 versus control.

a On study days 63 and 70 and at sacrifice in males, but not at 1000 mg/kg/day. b For study days 56–63 only in males, but not at 1000 mg/kg/day. c For study days 14–21 only in males.






F2 Offspring Toxicity Lactational survival (pnd 0–1) No toxicity Lactational body weight/litter (pnd 0) No toxicity Lactational sex ratio/litter No toxicity

pnd = postnatal day

2.6.7.14.B. Reproductive & Developmental Toxicity: Effects on Pre- & Post-Natal Development: Including Maternal Function

Report Title: TMC278-NC168: Oral (Gavage) Pre- and Post-natal Developmental Toxicity and Juvenile Toxicity Dose Range Finding Study in the Rat



2.6.7.14.B. TMC278-NC168: Oral (Gavage) Pre- and Post-natal Developmental Toxicity and Juvenile Toxicity Dose Range Finding Study in the Rat

Species/strain: Rat/Sprague Dawley Initial Age/weight: 10 weeks Duration of dosing: GD6-LD7 (dams); D12-D25 of age (pups) Day of mating: GD1

Administration route/method: Oral/Gavage Doses: 0, 40, 120, 400 mg base eq./kg/day TMC278.HCl Test article batch: Vehicle/formulation: 0.5% w/v HPMC.

Testing facility: Study No.: TMC278-NC168 GLP Compliance: No Date start of study: 20

No Observed Effect Level: 400 mg base eq./kg/day (maternal); 120 mg base eq./kg/day (fetal) Toxicokinetics: Maternal Dose (mg base eq./kg/day) 0 (vehicle) 0 (vehicle) 40 120 400 Pup Dose (mg base eq./kg/day) LD12-D25 0 (vehicle) 400 40 120 400 No. of Pups - - M: 8 F:8 M: 8 F:8 M: 8 F:8 M: 8 F:8 Died or killed prematurely - - 0 0 0 0 0 0 0 0 Cmax LD7 ( g/mL) exposure by lactation - - - - 0.05 0.07 0.08 0.08 0.16 0.14 Cmax D25 of age ( g/mL) oral gavage - - 6.4 4.9 2.6 5.8 3.7 3.6 9.1 7.3 AUC0-24h LD7 ( g.h/mL) exp. by lactation - - - - 0.62 0.74 0.94 0.91 1.9 1.8 AUC0-24h D25 of age ( g.h/mL) oral gavage - - 43 39 12 18 34 28 50 53 - not determined; F: female; M: male; - not determined; GD: gestation day; HPMC: hydroxypropylmethylcellulose; LD: lactation day.

2.6.7.14.B. Reproductive & Developmental Toxicity: Effects on Pre- & Post-Natal Development: Including Maternal Function

Report Title: TMC278-NC168: Oral (Gavage) Pre- and Post-natal Developmental Toxicity and Juvenile Toxicity Dose Range Finding Study in the Rat



Noteworthy Findings Dose (mg base eq./kg/day) 0 (vehicle) 0 (vehicle) 40 120 400 F0 Females: No. of Animals 6 6 6 6 6 No. Pregnant 6 6 6 6 6 No. died or sacrificed moribund 0 0 0 0 0 Clinical observations - - - - - Gestation body weight - - - - - Lactation body weight - - - - - Gestation food consumption - - - - - Lactation food consumption - - - - - Mean duration of gestation (days) - - - - - Total no of litters - - - - - Mean no of pups born - - - - - Mean live birth index - - - - - Total litter death during LD - - - - - Maternal Dose (mg base eq./kg/day) 0 (vehicle) 0 (vehicle) 40 120 400 Pup Dose (mg base eq./kg/day) LD12-D25 0 (vehicle) 400 40 120 400 F1 litters 8M/8F 8M/8F 8M/8F 8M/8F 8M/8F Number of pups born 76 88 65 76 74 Pup Mortalities LD12-D25 - -- 1 2 Pup clinical signs LD12-D25 Poor condition - - - - 2 Decreased activity - - - - 2 Pup body weight - - - - - Pup necropsy observations - - - - - D: day; HPMC: hydroxypropylmethylcellulose; LD: lactation day; - no noteworthy findings; F: female; M: male

2.6.7.14.C. Reproductive & Developmental Toxicity: Effects on Pre- & Post-Natal Development: Including Maternal Function

Report Title: TMC278-NC131: Oral (Gavage) Pre- and Post-natal Developmental Toxicity Study in the Rat



2.6.7.14.C. TMC278-NC131: Oral (Gavage) Pre- and Post-natal Developmental Toxicity Study in the Rat

Species/strain: Rat/Sprague-Dawley Initial Age/weight: 9-10 weeks Duration of dosing: GD6 – LD20 Day of mating: GD0

Administration route/method: Oral/gavage Doses: 0, 40, 120, 400 mg base eq./kg/day TMC278.HCl Test article batch: Vehicle/formulation: 0.5% w/v HPMC

Testing facility: Study No.: TMC278-NC131 GLP Compliance: Yes Date start of study: 20

No Observed Adverse Effect Level: 400 mg base eq./kg/day (maternal and fetal) Noteworthy Findings Dose (mg base eq./kg/day) 0 40 120 400 F0 Females: No. of Animals 25 25 25 25 No. died or sacrificed moribund 0 2 0 2 Clinical observations - - - - Gestation body weight - - - - Lactation body weight - - - - Gestation food consumption - - - - Lactation food consumption - - - - Macroscopic observations - - - - Mean duration of gestation (days) - - - - Mean no. implantations - - - - Dose (mg/kg/day) 0 40 120 400 F1 generation (pre-weaning) 25 25 25 23 Mean no of pups born - - - - Mean live birth index (%) 100 100 100 99.2* Mean viability index (%) 99.8 99.4 97.6* 93.5** Mean lactation index (%) - - - - * p 0.05; ** p 0.01 (Analysis of variance). GD: gestation day; HPMC: hydroxypropylmethylcellulose; LD: lactation day; - no noteworthy findings

2.6.7.14.C. Reproductive & Developmental Toxicity: Effects on Pre- & Post-Natal Development: Including Maternal Function

Report Title: TMC278-NC131: Oral (Gavage) Pre- and Post-natal Developmental Toxicity Study in the Rat



Noteworthy Findings Dose (mg base eq./kg/day) 0 40 120 400 F1 generation (post-weaning) 20 20 20 20 No. died or sacrificed moribund 0 0 0 0 Pup clinical observations - - - - Pup body weight - - - - Pup body weight gain - - - - Auditory acuity - - - - Locomotor coordination - - - - Sexual developmental observations - - - - Fertility/mating performance - - - - Macroscopic observations - - - - - no noteworthy findings.

2.6.7.14.D. Reproductive & Developmental Toxicity: Effects on Pre- & Post-Natal Development: Including Maternal Function

Report Title: Oral (Gavage) Developmental and Perinatal/Postnatal Reproduction Toxicity Study of GS-4331-05 (bis-POC PMPA) in Rats Including a Postnatal Behavioral/Functional Evaluation


* = p 0.05; ** = p 0.01; a = Terminal weights for control groups (means, at day 21, postpartum) are shown. For treatment groups, % differences from controls are shown; b = No deaths were related to test article. All deaths were related to intubation errors with the exception of the rat in control group, which had dystocia; c = Maternal absolute food consumptions for control groups (during gestation & lactation) are shown. For treatment groups, % differences from controls are shown; d = Excludes 1 rat which was scarified moribund day 22 gestation; e = Excludes 1 rat which was scarified moribund day 19 gestation. f = not considered test article related. CONFIDENTIAL Page 296 17AUG2010

2.6.7.14.D. R990202: Oral (Gavage) Developmental and Perinatal/Postnatal Reproduction Toxicity Study of GS-4331-05 (bis-POC PMPA) in Rats Including a Postnatal Behavioral/Functional Evaluation

Design Similar to ICH 4.1.2 Duration of Dosing: Treatment of females: From day 7 of presumed gestation to day 20 of lactation or day 24 of presumed gestation (in rats that did not deliver a litter)

Study No: R990202

Species. Strain: Rat, Crl:CD (SD)IGS BR VAF/Plus

Initial age: 59 days Day of mating: Day 0 GLP Compliance: Yes Date of First Dose: 20 Method of Administration: Oral gavage Special Features: None Vehicle Formulation: -Suspension vehicle NOEL: Fo Females. 50mg/kg Litters Culled/Not Culled: At weaning (day 21 lactation) 25 pups per sex, per group, where possible, were

randomly chosen for continued evaluation. All other pups were sacrificed. Developmental Toxicity: 150 mg/kg F1 General Toxicity: 50 mg/kg F1 Behavioural, reproductive & developmental: 150 mg/kg

Daily Dose (mg/kg) 0 Control 50 150 450 600

F0 Toxicokinetics: From study R990202-PK Females AUC (0- ) g.hr/mL (day 20 post partum) - 5.883 11.716 26.761 27.286 No. Evaluated 25 25 25 25 25 Mortality b 1 0 1 1 1 Nunmber died 0 0 1 0 0 Moribund sacrificed 1 0 0 1 1






F0 Clinical Observation Females Presumed Gestation (cont) Maximal Possible Incidence (days x

rats)/No of Rats per Group 561/25 556/25 559/25 562/25 569/25

Red Perioral Substance 0/0 1/1 37/14* 123/24** 124/25** Excess Salivation 0/0 0/0 7/5 12/10** 28/13** Urine Stained Abdominal

Fur 0/0 0/0 0/0 9/4* 13/8**

Ungroomed Coat 0/0 0/0 0/0 6/1 17/5** Lactation Maximal Possible Incidence (days x

rats)/No of Rats per Group 504/24 525/25 494/24 382/24 260/22

Dehydration 4/4 1/1 2/2 10/6 16/8** Emaciation 0/0 6/1 0/0 5/4* 12/7** Urine Stained Abdominal

Fur 4/2 6/3 8/3 45/9** 28/11**

Red of Dried Perioral Substance 0/0 0/0 3/3 9/8** 12/7** Ungroomed Coat 0/0 1/1 0/0 6/4* 11/7** Cold to Touch 0/0 0/0 0/0 0/0 9/4** Necropsy Observations Gestation/Lactation

Body Weight (%)a 327.8g 0 0 0 0






F0 Gestation/Food Consumption (%)c 23.8g/day 0 0 -12** -18** Females Lactation/Food Consumption (%)c 54.2g/day 0 0 -7%* -15** (Cont) Delivery Observations Females with Sperm 25 25 25 25 25 Pregnant Females (%) 25 (100) 25 (100) 24 (96) 24 (96) 23 (92) Females with Delivery (%) 24 (100) D 25 (100) 24 (100) 24 (100) 22 (100)e Mean Duration of Gestation Days

(SD) 23.0 (0.2) 23.0 (0.2) 23.2 (0.4) 23.3 (0.5)*f 23.3 (0.5)

F1 Litters Means Implantations per Delivered Litter

(SD) 16.3 (2.1) 15.5 (2.6) 15.2 (1.6) 15.4 (1.7) 15.5 (1.6)

Live Births per Litter (SD) 14.7(2.2) 14.4 (2.9) 14.2 (1.7) 13.8 (3.0) 12.8 (2.9) Stillbirths (SD) 0.2 (0.6) 0.2(0.4) 0 (0.2) 0.7 (2.3) 2.0 (2.8)* Survivors Day 4 pp (%)

Viability Index 99.4

351/353 99.2

357/360 99.1

338/341 80.8

256/317 53.9

152/282** Survivors at Weaning (%)

Lactation Index 98.6

346/351 98.0

350/357 98.1

319/325 95.0

230/242 97.4

148/152 Weight at Birth (g) 6.7 (0.5) 6.5 (0.5) 6.4 (0.4) 5.5 (0.7)** 5.1 (0.6)** Weight at Weaning g (SD) 37.4 (5.4) 35.9 (7.1) 35.4 (5.8) 27.1(3.6) ** 25.7 (2.5) ** M F M F M F M F M F Sex Ratio of Fetuses 49.9 50.1 46.5 53.5 52.4 47.6 48.0 52.0 47.0 53.0






F1 Litters

Clinical Observations (Total Frequency: Days x Pups)

(Cont) Cold to Touch 3/1 1/1 0/0 64/6 80/11** Pale 3/1 0/0 0/0 32/3 80/11** Not Nesting 0/0 0/0 0/0 0/0 31/4** Umbilical Hernia 0/0 0/0 0/0 28/4** 3/1 Necropsy Observations Incidence Pup Appearance

Normal (%) 295 (99.7) 298 (99.3) 269 (100) 169 (94.4) ** 97 (99)

Incidence Pup Liver Appearance Pale (%)

0 (0) 0 (0) 0 (0) 10 (5.6)* 0 (0)

Incidence Pup Testes Small & Flaccid (%)

0 (0) 0 (0) 0 (0) 0 (0) 1 (1.0)**

Kidney Weights g (mean (SD)

0.54 (0.08) 0.52 (0.09) 0.51(0.09) 0.40 (0.04) ** 0.36 (0.05)**

2.6.7.15. Studies in Juvenile Animals Test Article: Rilpivirine/Tenofovir disoproxil fumarate


2.6.7.15. Studies on Juvenile Animals For RPV (TMC278), studies in juvenile animals are described in Tabulated Summary 2.6.7.14.B. (rat) and 2.6.7.7.X (cynomolgus monkey). For TDF, studies in rhesus monkeys are described in Tabulated Summary 2.6.7.17.C.

2.6.7.16.A. Local Tolerance Test Article: Rilpivirine


2.6.7.16. Local Tolerance 2.6.7.16.A. Rilpivirine

Type of Study In vitro System

or Species/Strain Route/Method of Administration


Dose (Batch) (Reference/ Controls) Noteworthy Findings Study Number

In Vitro Tests Bovine corneal opacity -permeability eye irritation assay

In vitro NA NA 20% w/w TMC278.HCl in 0.9% NaCl ( ) (Reference: imidazole)

Increase in opacity and no increase in permeability. TMC278 considered a moderate eye irritant

TMC278-NC202

Phototoxicity - Balb/c mouse fibroblast assay

In vitro NA NA 0 (vehicle), 0.001 to 2.16 mg/L TMC278.HCl ( ) (positive control: Chlorpromazine) In presence and absence of UVA

TMC278.HCl was not phototoxic TMC278-NC188a

In Vivo Tests Primary skin irritation

Rabbit/New Zealand White

Gauze patch applied to intact skin for 4 hours

1M + 2F

0.5 g of TMC278.HCl ( )

No skin reactions, staining of skin or corrosive effects were observed. Classified as “non-irritant” to rabbit skin.

TMC278-NC159a

Local lymph node Mouse/CBA/ Ca/Ola/Hsd

Topical application (3 consecutive days)

4 5, 10 and 35% TMC278.HCl in an acetone:olive oil 4:1 ( ) (positive control: streptozotocin)

TMC278 has no sensitizing properties. TMC278-NC199

DMSO = dimethyl sulfoxide; F = female; M = male; NA = not applicable; UVA = ultraviolet A a GLP compliant

2.6.7.16.B. Local Tolerance Test Article: Tenofovir disoproxil fumarate


2.6.7.16.B. Tenofovir DF

Species Strain

Method of Administration.

Doses (mg/kg)

Gender and No. per Group Noteworthy Findings

Study Number


Topical conjunctival sac application

0.1 mL (0.0760g)

6 (3M, 3F) Very severe irritant to rabbit ocular tissues B990165


Topical dermal application

0.5 g 6M Slight irritant to rabbit skin B990166

Emtricitabine/Rilpivirine/Tenofovir Disoproxil Fumarate Section 2.6.7 Nonclinical Summary Final


2.6.7.17. Other Toxicity Studies 2.6.7.17.A. Emtricitabine

Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Immunotoxicity of FTC Rat/ Sprague-Dawley

Oral gavage 28 Days 0, 60, 240 and 1000

M: 5 and F: 5 The objective of the study was to assess the immunotoxicity of TP-0006 (FTC) in the rat following once daily oral gavage administration for 28 days. No adverse clinical signs were noted during the dosing period. Body weight was unaffected by treatment with TP-0006. There was no effect of FTC at dose levels up to 1000 mg/kg/day on the immune response to sheep red blood cells.

TOX146

Metabolites & Impurities Mouse, CD-1 (ICR)


20 (10M, 10F) The NOEL for oral gavage administration of TP-0006 test article (FTC produced by the chemistry method of synthesis) to mice for 28 consecutive days was 450 mg/kg/day, the highest dose tested.

TX-162-2001



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Mouse, CD-1 (ICR)

Oral gavage, TP-0006 and TP-0296

28 days (13 day post dose recovery)

0, 50/1, 150/3, 450/9

20 (10M, 10F) All mice survived to the scheduled necropsy. There were no observations related to treatment with the combination of TP-0006 (FTC) and TP-0296 at any dose level. Body weight, food consumption, hematology, serum biochemistry values and organ weights were unaffected by administration of the test article combination at any dose level. There were no gross or microscopic findings attributed to the test article combination. The no-observed-effect level (NOEL) for oral administration of TP-0006 in combination with TP-0296 was 450/9 mg/kg/day TP-0006/TP-0296, the highest dose combination tested.

TOX153

S typhimurium & E Coli Reverse Mutation Assay of TP-006 and TP-0296

In vitro - For TP-0006: 1.5 to 5000

g/plate. For TP-0296: 0.015 to 50

g/plate. Mutagenicity assay: For TP-0006: 50 to 5000 g/plate. For TP-0296: 0.50 to 50

g/plate

- Cytotoxic Effects: no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The maximum dose of TP-0006 (FTC) and TP-0296 tested were 5000 and 50 g per plate respectively. Neither precipitate nor appreciable toxicity was observed. Genotoxic Effects: No positive mutagenic responses were observed with any of the tester strains in either presence or absence of S9 activation. Mutagenic responses were observed with the positive control articles.

TOX151



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Chromosome aberration in CHO-K1 cells

In vitro - 0.5 to 5000 g/mL with 1% TP-0296

- Cytotoxic Effects: Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was not observed at any dose level in both the non-activated and S9 activated 4 hour exposure groups, or in the non-activate 20 hour continuous exposure group. Based upon the results of the toxicity study, the dose levels selected for testing in the chromosome aberration assay were ranging from 313 to 5000 g/mL for TP-0006 (FTC) and from 3.13 to 50 g/mL for TP-0296. Genotoxic Effects: Based on the result of this study, it was concluded that TP-0006 with 1% TP-0296 was negative for the induction of structural and numerical chromosome aberrations in CHO cells.

TOX152







Duration of Dosing Batch No.

Doses (mg base eq./kg/day) Noteworthy Findings

Study Number

Mechanistic Studies (RPV) Dog/Beagle (endocrinology study) 6M/group


Single dose

0 (vehicle), 20, or 80 TMC278 base

No mortality. No relevant clinical signs. No effects on body weight, cortisol, ACTH, aldosterone and CRF. 20 mg/kg: Cmax: 2.8 g/mL; AUC0- : 87 g.h/mL 80 mg/kg: Cmax: 2.8 g/mL; AUC0 :105 g.h/mL.

TMC278-Exp5627

Guinea Pig Adrenal Cell Test (Evaluation of cortisol biosynthesis)

In vitro (DMSO 1%)

-

10-9 to 10-5M (0.37 ng/mL to 3.7 g/mL) TMC278 base. Positive control: Etomidate (10-9 to 10-5 M)

TMC278 inhibited cortisol synthesis in one experiment with an IC50 value of 6 M but showed no significant inhibition in the second experiment. Etomidate rendered an IC50 value of 0.5 nM

TMC278-Exp5653

Dog adrenal cortex (Evaluation of the cortisol in crude subcellular fraction)

In vitro -

0.03 to 75 M (0.011 to 27.75 g/mL) TMC278 base Positive control: Etomidate (0.28 M)

75 M: 39% inhibition of the conversion of pregnenolone. Progesterone and 17- OH-progesterone concentrations increased with increasing concentrations of TMC278 whereas 11-deoxycorticosterone, 11-deoxycortisol, and corticosterone concentrations decreased with increasing concentrations of TMC278. No changes in cortisol. Etomidate showed a 38% inhibition of the corticosteroid synthesis.

TMC278-FK4790

a Unless indicated differently; ACTH: adreno corticotropic hormone ; CA: citric acid; CRF: corticotrophin releasing factor; DMSO: dimethylsufoxide; F: female; M: male; PEG400: polyethylene glycol 400.







Doses (mg/kg/day) Noteworthy Findings

Study Number

Impurities (RPV) Qualification of and according to ICH Q3A S. typhimurium TA98, TA100, TA1535, TA1537, TA102 +/- S9

In vitro (DMSO)

-

(TMC278HCl) +

( )

0 (vehicle), 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 g/plate TMC278.HCl spiked with 4%

Precipitation at 55.69 g/mL and above. No evidence of mutagenic properties/

TMC278-NC308a

S. typhimurium TA98, TA100, TA1535, TA1537, TA102 +/- S9

In vitro (DMSO)

-

(TMC278HCl) +

( )

0 (vehicle), 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 g/plate TMC278.HCl spiked with 4%

Precipitation at 100 g/plate and above/ No evidence of mutagenic properties/

TMC278-NC309a

a GLP compliant; DMSO: dimethyl sulfoxide; Exp: experiment.


不純物B*

不純物B*

不純物C*

不純物C*









Study Number

and (continued) Mouse lymphoma L5178Y/tk+/-

In vitro (DMSO)

-

(TMC278HCl) +

( )

Exp 1: 0 (vehicle), 2.5, 5, 10, 15, 20, 25, 30, 40 g/mL 3 hours + S9 3 hours – S9 Exp 2: 0 (vehicle), 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 50, 75 g/mL. 3 hours +S9 24 hours –S9 TMC278 HCl spiked with 4%

Precipitation at 15 g/mL and above/ No evidence of mutagenic or clastogenic properties.

TMC278-NC311a

a GLP compliant; DMSO: dimethyl sulfoxide; Exp: experiment.

不純物C*不純物B*

不純物B*

不純物B*









Study Number

Qualification of and according to ICH Q3A (continued) Mouse lymphoma L5178Y/tk+/-

In vitro (DMSO)

-

(TMC278HCl) +

( )

Exp 1:0 (vehicle), 2.5, 5, 10, 15, 20, 25, 30 g/mL 3 hours + S9 3 hours – S9 Exp 2: 0 (vehicle), 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 50, 75 g/mL. 3 hours +/-S9 24 hours –S9 TMC278 HCl spiked with 4%

Precipitation at 15 g/mL and above. No mutagenic or clastogenic properties. .

TMC278-NC312a

Rat/Sprague Dawley 10M and 10F/group


1-month

+

( ) Or

+

( )

0 (vehicle), 10 TMC278 HCl, 10 TMC278 HCl spiked with 4%

, 10 TMC278 HCl spiked with 4%

.

No effects in any group different from the negative control group.

TMC278-NC314a

a GLP compliant; DMSO: dimethyl sulfoxide; F: female; HPMC: hydroxypropyl methylcellulose; M: male.

不純物B*

不純物B* 不純物B*

不純物C*

不純物C*

不純物C*

不純物C*

不純物C*







Duration of Dosing Batch no.

Doses (mg/kg/day)

Noteworthy Findings Study No./

Location in CTD


In vitro (DMSO)

--- Exp 1and 2: 0 (vehicle), 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000

Increased mutation frequency in strains TA98 and TA100 in the presence of rat liver S9-mix.

TMC278-NC179a


In vitro (DMSO)

--- Exp 1, 2, 3, 4, 5 and 6: 0 (vehicle), 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000

Increasede mutation frequency in strain TA1537 in the absence of S9-mix and in strain TA98 in the presence of S9-mix.

TMC278-NC165


In vitro (DMSO)

---

Exp 1 (2): 0 (0), 78.13 (93.75), 156.25 (187.5), 312.5 (375), 625 (750), 1250 (1500), 2500 (3000) and 5000 (5000)

Increased mutation frequency in the absence and the presence of S9-mix in TA98 and TA1537 and in the presence of S9-mix in TA100.

TMC278- 1646_0016315a

a GLP compliant; DMSO: dimethyl sulfoxide

不純物D'*

不純物D'*

不純物D'*

不純物D'*








Doses (mg/kg/day)

Noteworthy Findings Study No./

Location in CTD

Human lymphocytes +/- S9

In vitro/ (DMSO)

4 and 24 hours exposure

Not reported 0 (vehicle), 65.22, 130.44, 260.88 and 521.75 g/mL

Statistically significant increase in the frequency of cells with aberrations in the absence of metabolic activation (S9) after 4 hours exposure group at 521.75 μg/mL.

TMC278-NC184


In vitro (DMSO)

- Exp 1 (2): 0 (0), 78.13 (93.75), 156.25 (187.5), 312.5 (375), 625 (750), 1250 (1500), 2500 (3000) and 5000 (5000)

No evidence of mutagenic properties with any of the tested strains both in the absence and the presence of S9-mix.

TMC278- 1646_0015298a


In vitro (DMSO)

- Exp 1 (2): 0 (0), 78.13 (93.75), 156.25 (187.5), 312.5 (375), 625 (750), 1250 (1500), 2500 (3000) and 5000 (5000) Exp 3: 0, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000

No evidence of mutagenic properties with any of the tested strains both in the absence and the presence of S9-mix.

TMC278- 1646_0015299 a

a GLP compliant; DMSO: dimethyl sulfoxide

不純物D'*

不純物E*

不純物F*




2.6.7.17.C. Tenofovir DF

Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Antigenicity of TDF Guinea Pig, Hartley

Topical 5 weeks: 3 wks induction, 1 wk challenge & 1 wk re-challenge

75% w/v q7d, for 3 wks, 50% w/v or 100% for challenge & 65% w/v for re-challenge.

10M, 10F Not a contact skin sensitizer in guinea pigs. G990167

Mechanistic Studies (TDF) Human osteoblasts-like (NHOst) cells

In vitro 3 weeks 10 μg/mL - Three week continuous in vitro exposure of NHOst cells to tenofovir (10 g/mL) was not cytotoxic and did not alter their cellular function of calcium deposition.

V2000122



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Rat, Sprague Dawley

Oral/Gavage & Intraperitoneal (IP)

3 days 0, 400 6M The daily administration of tenofovir DF at a dose of 400 mg/kg/day by oral gavage for 3 days to 2 groups of rats caused statistically significant reductions in serum phosphorus ( 8 to 12%) and statistically significant reductions in urinary phosphorus excretion ( 90%). Oral phosphate supplementation to one tenofovir DF-treated group and the gavage vehicle group and intraperitoneal (IP) phosphate supplementation to the other tenofovir DF- and vehicle-treated groups following the third dose of either vehicle or tenofovir DF dose serum phosphorus concentrations to approximate control values. Urinary phosphorus excretion of the tenofovir DF-treated group that received IP phosphate supplementation to approximate both control group values. Urinary phosphorus excretion of the tenofovir DF-treated group that received phosphate supplementation by oral gavage also but was only ~1/3 the response of control groups. The findings suggest that the administration of tenofovir DF by oral gavage impaired the intestinal absorption of phosphate resulting in renal retention of phosphate. Phosphate supplementation administered by oral gavage appeared to at least partially overcome the impairment while the urinary phosphate excretion of the tenofovir DF-treated group that received IP phosphate supplementation indicated normal renal function for phosphate excretion.

R2000095



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Rat, Sprague Dawley

Oral/Gavage or Intravenous (IV)

3 days 0, Tenofovir: 50 (IV), 180 (PO) TDF: 400 (PO)

6M Tenofovir DF (oral): Marked urinary phosphorus excretion, slight serum phosphorus. Tenofovir (IV): slight serum phosphorus, no effect on urinary phosphorus excretion. Tenofovir (oral): no effect on serum phosphorus or urinary phosphorus excretion. Four hours after oral phosphate supplementation:

in urinary phosphorus excretion for all groups of similar magnitude except for the tenofovir DF group in which the was ~1/3 the of the other groups, slight serum phosphorus in all groups compared to pre-phosphate values with the tenofovir DF group having the greatest increase. All serum and urine values approached either prestudy or pre-phosphate supplementation values by the end of the study. The findings suggest that the oral administration of tenofovir DF, the pro-drug of tenofovir, impaired intestinal phosphate absorption with resulting renal phosphate absorption. Oral phosphate supplementation appeared to overcome the impairment resulting in urinary phosphate excretion. Neither tenofovir administered orally or intravenously altered urinary phosphorus excretion before or after phosphate supplementation.

R2000099



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Rat, Sprague Dawley

Oral/Gavage 6 days 0, 400 8M Objective: examine the effect of daily treatment of tenofovir DF on serum or plasma calcium and phosphorus; urinary excretion of calcium, phosphorus, cAMP, and NAG; and biochemical markers of bone remodeling in rats. Slight serum or plasma calcium, ionized calcium and phosphorus, serum 25-vitamin D3, serum 1,25 vitamin D3, parathyroid hormone unchanged on day 6. Urinary calcium excretion was initially on days 0, 1, and 2 and then approximated control values for the remainder of the study, marked urinary phosphorus excretion throughout the study, NAG excretion, urinary cAMP excretion unchanged. Microscopically: renal tubular epithelial karyomegaly.

R2000043

Rat Sprague Dawley

Oral/Gavage 28 days 0 (adefovir dipivoxil ADV, alone), 300 TDF alone & 300 TDF + 40 ADV combination group.

6M For both ADV alone and combination treated groups mean body weights , BUN & creatinine and glucosuria, proteinuria, calciuria & renal tubular nephrosis observed; severity in ADV alone group. Hypophosphaturia seen in TDF alone & combination groups. Combination & ADV alone groups has proliferative & degenerative changes in the intestine; while group with TDF alone had slight proliferative changes & thickening of intestinal mucosa. All groups had AST & ALT through day 14, however there were no histologic correlated to these changes. Combination group mean serum lactate , however because there were no changes in cytochrome c oxidase, citrate synthase, or mtDNA content in liver, kidney or skeletal muscle these changes were most likely not caused by mitochondrial dysfunction.

R2000096



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Rat, Sprague Dawley

Oral/Gavage 28 days 0, 40, 400 10M (TK cohort:

18M/low dose group and 21M/high

dose group)

400 mg/kg/d: body weights/body weight gain. 40 and 400 mg/kg/day: dose-related osteopenia ( bone mineral content and bone mineral density based on histomorphometry and bone densitometry, respectively). Microscopic indices of bone formation and mineralization were unaffected. There were no histologic lesions in the decalcified sections of distal femur from either tenofovir DF-treated group. 400 mg/kg/d: Slight serum calcium, moderate urinary calcium excretion, marked urinary phosphorus excretion. Renal tubular epithelial karyomegaly.

R2000036

Woodchuck (Marmota monax)

Oral/Gavage 90 days 0, 15, 50 18 (9M, 9F) No evidence of mitochondrial toxicity was observed W2000042



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Monkey, Rhesus Oral gavage & SC 56 days TDF 0, 30, 250, 600, PO tenofovir, 30 SC.

6 (3M, 3F) 600 mg TDF/kg/d: 2 monkeys developed severe renal toxicity ~Day 20 and the group euthanized; BUN and creatinine in 600 mg/kg/d TDF group and 30 mg/d sc tenofovir group. The daily administration of TDF orally or tenofovir sc resulted in dose-dependent in serum phosphorus through 4 weeks. Hyperphosphaturia was noted for high dose TDF and tenofovir sc. Phosphate supplementation (after D 29) normalized serum phosphate and fractional excretion of phosphate. Slight to moderate in ALT and AST occurred in treated animals. Histological changes in renal tubular epithelial cells (250, 600 mg/kg/d TDF, 30 mg/kg/d tenofovir sc) included karyomegaly, generalised swelling, fine basophilic granulation, individual cell necrosis, and mitotic cells. Histopathological changes in the testes of animals from all treatment groups were observed. The significance of this alteration is hampered by the small number of animals. There were minimal changes in biochemical markers of bone alterations and no alterations in bone morphology. No evidence that TDF caused mitochondrial injury at doses up to 250 mg/kg/d. In summary: TDF administered by nasogastric gavage and tenofovir administered by subcutaneous injection resulted in a reduction of serum phosphorus that was normalized by phosphate supplementation administered by nasogastric gavage.

P2000078



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Metabolites & Impurities

Rat, Sprague Dawley


20 (10M, 10F)

Toxicokinetics 12 (6M, 6F)

No treatment-related findings were observed with either TDF or degraded TDF. No differences between TDF & degraded TDF were noted. NOEL: Lot # : 300 mg/kg/d; lot#

: 300 mg/kg/d. There were no treatment-related findings for either lot.

R2000081

Genotoxicity S typhimurium & E Coli Mammalian Microsome Reverse Mutation Assay of Tenofovir

In vitro - 92.2 to 4610 g/plate +/-

metabolic activation & 100 to 5000

g/plate – metabolic activation

- Positive increases in the mean number of revertants with only tester strain TA1535 in the presence of S9.

95-TOX-1278-006

L5178Y TK +/- Mouse lymphoma Forward Mutation Assay of Tenofovir

In vitro - 9.77 to 5000 g/mL

- Tenofovir was negative without activation and equivocal with activation.

95-TOX-1278-007



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Safety Studies in Efficacy Models (TDF) Monkey, Rhesus Tenofovir,

subcutaneous (SC) Gestational Day 80–157; Infants continued through 9 months

0, 30 6 (infected) 12 maternal/fetal

pairs (6 non-infected, 6 infected)

Decreased infant body weights and alterations of select serum biochemical parameters (e.g., decreased phosphorus levels, elevated alkaline phosphatase) were shown to occur in 67% of tenofovir-treated infants, with severe growth restriction and bone-related toxicity in approximately 25% of animals studied. These data suggest there is the potential for bone-related toxicity at chronic, high dosages, particularly in infants.

J Acquir Immune Defic

Syndr Hum Retrovirol

1999 Apr 1;20 (4):323-33.

{1787}

Monkey, Rhesus Tenofovir, subcutaneous (SC)

28 days and at least 434 days

0, 30 5 No behavioural signs (e.g., reluctance to stand, abnormal spine curvature) of bone toxicity were observed in animals treated with tenofovir for 28 days. After 350 days of tenofovir treatment, serum phosphorous , mean alkaline phosphatase activity , serum Ca no change; 1/5 animals bone density (L3-L5) and displacement (T4-T5) . 4/5 animals had bone loss in the spine or pelvis.

T1278-00034



Species Strain



Duration of Dosing

Doses (mg/kg)


Study Number

Monkey, Rhesus Tenofovir, subcutaneous (SC)

Short-term treatment: 1 day to 12 weeks. Prolonged administration with high dose: 4 month to 25 months. Prolonged low dose treatment:

5 years.

Short term treatment: 4, 10, 30 Prolonged administration with high dose:30 with dose reductions to. 10, 5, 2.5 Prolonged low dose treatment: 10

Short-term treatment: 39 animals. Prolonged administration with high dose: 15 animals. Prolonged low dose treatment: 3 animals.

Short-term treatment & prolonged low dose treatment: No adverse events on health or growth. Prolonged administration with high dose: Clearance tenofovir , Fanconi-like syndrome, with glucosuria & hypophosphataemia, bone demineralization, growth plate deformations & growth restrictions all observed. Renal effects largely reversible on tenofovir withdrawal or dose reduction to 5–10 mg/kg.

{7311}, P2000124

Monkey, Rhesus Tenofovir, subcutaneous (SC) Followed by In vitro analysis of bone specimens from different studies and experimental regimens

Up to 2 years 10, 30 & 30 (with dose reduction to 10)

9-14 per group (M & F)

50% of animals treated with tenofovir 30 mg/kg for at least 4 months had increased osteoid seam widths & osteomalacia. Bones from treatment discontinued or dose reduced treated animals (10 mg/kg) were normal suggesting potential reversal of bone changes.

T1278-00030



2.6.7.18. References

1787 Tarantal A, Marthas ML, Shaw J-P, Cundy KC, Bischofberger N. Administration of 9-[2-(R) (phosphonomethoxy)propyl]adenine (PMPA) to gravid and infant rhesus macaques (Macaca mulatta): safety and efficacy studies. J Acquir Immune Defic Syndr Hum Retrovirol 1999 Apr 1;20 (4):323-33.

7311 Van Rompay KKA, Brignolo LL, Meyer DJ, Jerome C, Tarara R, Spinner A, et al. Biological effects of short-term or prolonged administration of 9-[2-(phosphonomethoxy)propyl]adenine (tenofovir) to newborn and infant rhesus macaques. Antimicrob Agents Chemother 2004 May;48 (5):1469-87.

section 2.6 nonclinical summary section 2.6.6 toxicology … · 2015-05-29 · toxicology...

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